Modulators of cellular proliferation

ABSTRACT

The present invention relates to regulation of cellular proliferation. More particularly, the present invention is directed to nucleic acids encoding protein kinase C ζ (PKC-ζ), phospholipase C-β1 (PLC-β1), protein tyrosine kinase 2 (FAK), protein tyrosine kinase 2b (FAK2), casein kinase 2 (CK2), cMET tyrosine kinase (cMET), flap structure specific endonuclease 1 (FEN1), REV1 dCMP transferase (REV1), apurinic/apyrimidinic nuclease 1 (APE1), cyclin dependent kinase 3 (CDK3), PIM1 kinase (PIM1), cell division cycle 7 kinase (CDC7L1), cyclin dependent kinase 7 (CDK7), cytokine inducible kinase (CNK), potentially prenylated protein tyrosine phosphatase (PRL-3), serine threonine kinase 2 (STK2) or (NEK4), cyclin dependent serine threonine kinase (NKIAMRE), or histone acetylase (HBO1), which are involved in modulation of cell cycle arrest. The invention further relates to methods for identifying and using agents, including small molecule chemical compositions, antibodies, peptides, cyclic peptides, nucleic acids, RNAi, antisense nucleic acids, and ribozymes, that modulate cell cycle arrest via modulation of protein kinase C ζ (PKC-ζ), phospholipase C-β1 (PLC-β1), protein tyrosine kinase 2 (FAK), protein tyrosine kinase 2b (FAK2), casein kinase 2 (CK2), cMET tyrosine kinase (cMET), flap structure specific endonuclease 1 (FEN1), REV1 dCMP transferase (REV1), apurinic/apyrimidinic nuclease 1 (APE1), cyclin dependent kinase 3 (CDK3), PIM1 kinase (PIM1), cell division cycle 7 kinase (CDC7L1), cyclin dependent kinase 7 (CDK7), cytokine inducible kinase (CNK), potentially prenylated protein tyrosine phosphatase (PRL-3), serine threonine kinase 2 (STK2) or (NEK4), cyclin dependent serine threonine kinase (NKIAMRE), or histone acetylase (HBO1), as well as to the use of expression profiles and compositions in diagnosis and therapy related to cell cycle regulation and modulation of cellular proliferation, e.g., for treatment of cancer and other diseases of cellular proliferation.

CROSS-REFERENCES TO RELATED APPLICATIONS

[0001] This application claims the benefit of provisional U.S.Application No. 60/395,443, filed Jul. 12, 2002, which is hereinincorporated by reference for all purposes.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

[0002] Not applicable.

FIELD OF THE INVENTION

[0003] The present invention relates to regulation of cellularproliferation. More particularly, the present invention is directed tonucleic acids encoding protein kinase C ζ (PKC-ζ), phospholipase C-β1(PLC-β1), protein tyrosine kinase 2 (FAK), protein tyrosine kinase 2b(FAK2), casein kinase 2 (CK2 or CK2α), cMET tyrosine kinase (cMET), flapstructure specific endonuclease 1 (FEN1), REV1 dCMP transferase (REV1),apurinic/apyrimidinic nuclease 1 (APE1), cyclin dependent kinase 3(CDK3), PIM1 kinase (PIM1), cell division cycle 7 kinase (CDC7L1),cyclin dependent kinase 7 (CDK7), cytokine inducible kinase (CNK),potentially prenylated protein tyrosine phosphatase (PRL-3), serinethreonine kinase 2 (STK2) or (NEK4), cyclin dependent serine threoninekinase (NKIAMRE), or histone acetylase (HBO1), which are involved inmodulation of cell cycle arrest. The invention further relates tomethods for identifying and using agents, including small moleculechemical compositions, antibodies, peptides, cyclic peptides, nucleicacids, RNAi, antisense nucleic acids, and ribozymes, that modulate cellcycle arrest via modulation of protein kinase C ζ (PKC-ζ), phospholipaseC-β1 (PLC-β1), protein tyrosine kinase 2 (FAK), protein tyrosine kinase2 b (FAK2), casein kinase 2 (CK2 or CK2α), cMET tyrosine kinase (cMET),flap structure specific endonuclease 1 (FEN1), REV1 dCMP transferase(REV1), apurinic/apyrimidinic nuclease 1 (APE1), cyclin dependent kinase3 (CDK3), PIM1 kinase (PIM1), cell division cycle 7 kinase (CDC7L1),cyclin dependent kinase 7 (CDK7), cytokine inducible kinase (CNK),potentially prenylated protein tyrosine phosphatase (PRL-3), serinethreonine kinase 2 (STK2) or (NEK4), cyclin dependent serine threoninekinase (NKIAMRE), or histone acetylase (HBO1), as well as to the use ofexpression profiles and compositions in diagnosis and therapy related tocell cycle regulation and modulation of cellular proliferation, e.g.,for treatment of cancer and other diseases of cellular proliferation.

BACKGROUND OF THE INVENTION

[0004] Cell cycle regulation plays a critical role in neoplasticdisease, as well as disease caused by non-cancerous, pathologicallyproliferating cells. Normal cell proliferation is tightly regulated bythe activation and deactivation of a series of proteins that constitutethe cell cycle machinery. The expression and activity of components ofthe cell cycle can be altered during the development of a variety ofhuman disease such as cancer, cardiovascular disease, psoriasis, whereaberrant proliferation contributes to the pathology of the illness.There are genetic screens to isolate important components for cell cycleregulation using different organisms such as yeast, worms, flies, etc.However, involvement of a protein in cell cycle regulation in a modelsystem is not always indicative of its role in cancer and otherproliferative disease. Thus, there is a need to establish screening forunderstanding human diseases caused by disruption of cell cycleregulation. Identifying proteins, their ligands and substrates, anddownstream signal transduction pathways involved in cell cycleregulation and neoplasia in humans is important for developingtherapeutic regents to treat cancer and other proliferative diseases.

BRIEF SUMMARY OF THE INVENTION

[0005] The present invention therefore provides nucleic acids encodingprotein kinase C ζ (PKC-ζ), phospholipase C-β1 (PLC-β1), proteintyrosine kinase 2 (FAK), protein tyrosine kinase 2b (FAK2), caseinkinase 2 (CK2 or CK2α), cMET tyrosine kinase (cMET), flap structurespecific endonuclease 1 (FEN1), REV1 dCMP transferase (REV1),apurinic/apyrimidinic nuclease 1 (APE1), cyclin dependent kinase 3(CDK3), PIM1 kinase (PIM1), cell division cycle 7 kinase (CDC7L1),cyclin dependent kinase 7 (CDK7), cytokine inducible kinase (CNK),potentially prenylated protein tyrosine phosphatase (PRL-3), serinethreonine kinase 2 (STK2) or (NEK4), cyclin dependent serine threoninekinase (NKIAMRE), or histone acetylase (HBO1), which are involved inmodulation of cell cycle arrest in tumor cells and other pathologicallyproliferating cells. The invention therefore provides methods ofscreening for compounds, e.g., small organic molecules, antibodies,peptides, cyclic peptides, nucleic acids, antisense molecules, RNAi, andribozymes, that are capable of modulating cellular proliferation and/orcell cycle regulation, e.g., either inhibiting cellular proliferation,or activating apoptosis. Therapeutic and diagnostic methods and reagentsare also provided. Modulators of protein kinase C ζ (PKC-ζ),phospholipase Cβ1 (PLC-β1), protein tyrosine kinase 2 (FAK), proteintyrosine kinase 2b (FAK2), casein kinase 2 (CK2 or CK2α), cMET tyrosinekinase (cMET), flap structure specific endonuclease 1 (FEN1), REV1 dCMPtransferase (REV1), apurinic/apyrimidinic nuclease 1 (APE1), cyclindependent kinase 3 (CDK3), PIM1 kinase (PIM1), cell division cycle 7kinase (CDC7L1), cyclin dependent kinase 7 (CDK7), cytokine induciblekinase (CNK), potentially prenylated protein tyrosine phosphatase(PRL-3), serine threonine kinase 2 (STK2) or (NEK4), cyclin dependentserine threonine kinase (NKIAMRE), or histone acetylase (HBO1) aretherefore useful in treatment of cancer and other proliferativediseases.

[0006] One embodiment of the present invention provides a method foridentifying a compound that modulates cell cycle arrest. A cellcomprising a protein kinase C ¢ (PKC-ζ), phospholipase C-β1 (PLC-β1),protein tyrosine kinase 2 (FAK), protein tyrosine kinase 2b (FAK2),casein kinase 2 (CK2 or CK2α), cMET tyrosine kinase (cMET), flapstructure specific endonuclease 1 (FEN1), REV1 dCMP transferase (REV1),apurinic/apyrimidinic nuclease 1 (APE1), cyclin dependent kinase 3(CDK3), PIM1 kinase (PIM1), cell division cycle 7 kinase (CDC7L1),cyclin dependent kinase 7 (CDK7), cytokine inducible kinase (CNK),potentially prenylated protein tyrosine phosphatase (PRL-3), serinethreonine kinase 2 (STK2) or (NEK4), cyclin dependent serine threoninekinase (NKIAMRE), or histone acetylase (HBO1) polypeptide or fragmentthereof is contacted with the compound. The protein kinase C ζ (PKC-ζ),phospholipase C-β1 (PLC-β1), protein tyrosine kinase 2 (FAK), proteintyrosine kinase 2b (FAK2), casein kinase 2 (CK2 or CK2α), cMET tyrosinekinase (cMET), flap structure specific endonuclease 1 (FEN1), REV1 dCMPtransferase (REV1), apurinic/apyrimidinic nuclease 1 (APE1), cyclindependent kinase 3 (CDK3), PIM1 kinase (PIM1), cell division cycle 7kinase (CDC7L1), cyclin dependent kinase 7 (CDK7), cytokine induciblekinase (CNK), potentially prenylated protein tyrosine phosphatase(PRL-3), serine threonine kinase 2 (STK2) or (NEK4), cyclin dependentserine threonine kinase (NKIAMRE), or histone acetylase (HBO1)polypeptide or fragment thereof may be encoded by a nucleic acid thathybridizes under stringent conditions to a nucleic acid encoding apolypeptide having an amino acid sequence of SEQ ID NO:2, 4, 6, 8, 10,12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, or 36. The chemical orphenotypic effect of the compound upon the cell comprising the proteinkinase C ζ (PKC-ζ), phospholipase C-β1 (PLC-1), protein tyrosine kinase2 (FAK), protein tyrosine kinase 2b (FAK2), casein kinase 2 (CK2 orCK2α), cMET tyrosine kinase (cMET), flap structure specific endonuclease1 (FEN1), REV1 dCMP transferase (REV1), apurinic/apyrimidinic nuclease 1(APE1), cyclin dependent kinase 3 (CDK3), PIM1 kinase (PIM1), celldivision cycle 7 kinase (CDC7L1), cyclin dependent kinase 7 (CDK7),cytokine inducible kinase (CNK), potentially prenylated protein tyrosinephosphatase (PRL-3), serine threonine kinase 2 (STK2) or (NEK4), cyclindependent serine threonine kinase (NKIAMRE), or histone acetylase (HBO1)polypeptide or fragment thereof is determined, thereby identifying acompound that modulates cell cycle arrest. The chemical or phenotypiceffect may be determined by measuring enzymatic activity of the proteinkinase C ζ (PKC-ζ), phospholipase C-β1 (PLC-β1), protein tyrosine kinase2 (FAK), protein tyrosine kinase 2b (FAK2), casein kinase 2 (CK2 orCK2α), cMET tyrosine kinase (cMET), flap structure specific endonuclease1 (FEN1), REV1 dCMP transferase (REV1), apurinic/apyrimidinic nuclease 1(APE1), cyclin dependent kinase 3 (CDK3), PIM1 kinase (PIM1), celldivision cycle 7 kinase (CDC7L1), cyclin dependent kinase 7 (CDK7),cytokine inducible kinase (CNK), potentially prenylated protein tyrosinephosphatase (PRL-3), serine threonine kinase 2 (STK2) or (NEK4), cyclindependent serine threonine kinase (NKIAMRE), or histone acetylase (HBO1)polypeptide. The chemical or phenotypic effect may be determined bymeasuring cell cycle arrest. The cell cycle arrest may be measured byassaying DNA synthesis or fluorescent marker level. DNA synthesis may bemeasured by 3H thymidine incorporation, BrdU incorporation, or Hoeschtstaining. The fluorescent marker may be a cell tracker dye or greenfluorescent protein. Modulation may be activation of cell cycle arrestor activation of cancer cell cycle arrest. The host cell may be a cancercell. The cancer cell may be a breast, prostate, colon, or lung cancercell. The cancer cell may be a transformed cell line, such as, forexample, PC3, H1299, MDA-MB-231, MCF7, A549, or HeLa. The cancer cellmay be p53 null, p53 mutant, or p53 wild-type. The polypeptide mayrecombinant. The polypeptide may be encoded by a nucleic acid comprisinga sequence of SEQ ID NO:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25,27, 29, 31, 33, or 35. The compound may be an antibody, an antisensemolecule, a small organic molecule, a peptide, a circular peptide, or ansiRNA molecule.

[0007] Another embodiment of the invention provides a method foridentifying a compound that modulates cell cycle arrest. The compound iscontacted with a protein kinase C ζ(PKC-ζ), phospholipase C-β1 (PLC-β1),protein tyrosine kinase 2 (FAK), protein tyrosine kinase 2b (FAK2),casein kinase 2 (CK2 or CK2α), cMET tyrosine kinase (cMET), flapstructure specific endonuclease 1 (FEN1), REV1 dCMP transferase (REV1),apurinic/apyrimidinic nuclease1 (APE1), cyclin dependent kinase 3(CDK3), PIM1 kinase (PIM1), cell division cycle 7 kinase (CDC7L1),cyclin dependent kinase 7 (CDK7), cytokine inducible kinase (CNK),potentially prenylated protein tyrosine phosphatase (PRL-3), serinethreonine kinase 2 (STK2) or (NEK4), cyclin dependent serine threoninekinase (NKIAMRE), or histone acetylase (HBO1) polypeptide or fragmentthereof. The protein kinase C ζ (PKC-ζ), phospholipase C-β1 (PLC-β1),protein tyrosine kinase 2 (FAK), protein tyrosine kinase 2b (FAK2),casein kinase 2 (CK2 or CK2α), cMET tyrosine kinase (cMET), flapstructure specific endonuclease 1 (FEN1), REV1 dCMP transferase (REV1),apurinic/apyrimidinic nuclease 1 (APE1), cyclin dependent kinase 3(CDK3), PIM1 kinase (PIM1), cell division cycle 7 kinase (CDC7L1),cyclin dependent kinase 7 (CDK7), cytokine inducible kinase (CNK),potentially prenylated protein tyrosine phosphatase (PRL-3), serinethreonine kinase 2 (STK2) or (NEK4), cyclin dependent serine threoninekinase (NKIAMRE), or histone acetylase (HBO1) polypeptide or a fragmentthereof may be encoded by a nucleic acid that hybridizes under stringentconditions to a nucleic acid encoded by a polypeptide comprising anamino acid sequence of SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22,24, 26, 28, 30, 32, 34, or 36. The physical effect of the compound uponthe protein kinase C ζ (PKC-ζ, phospholipase C-β1 (PLC-β1), proteintyrosine kinase 2 (FAK), protein tyrosine kinase 2b (FAK2), caseinkinase 2 (CK2), cMET tyrosine kinase (cMET), flap structure specificendonuclease 1 (FEN1), REV1 dCMP transferase (REV1),apurinic/apyrimidinic nuclease 1 (APE1), cyclin dependent kinase 3(CDK3), PIM1 kinase (PIM1), cell division cycle 7 kinase (CDC7L1),cyclin dependent kinase 7 (CDK7), cytokine inducible kinase (CNK),potentially prenylated protein tyrosine phosphatase (PRL-3), serinethreonine kinase 2 (STK2) or (NEK4), cyclin dependent serine threoninekinase (NKIAMRE), or histone acetylase (HBO1) polypeptide is determined.The chemical or phenotypic effect of the compound upon a cell comprisinga protein kinase C ζ (PKC-ζ), phospholipase C-β1 (PLC-β1), proteintyrosine kinase 2 (FAK), protein tyrosine kinase 2b (FAK2), caseinkinase 2 (CK2 or CK2α), cMET tyrosine kinase (cMET), flap structurespecific endonuclease 1 (FEN1), REV1 dCMP transferase (REV1),apurinic/apyrimidinic nuclease 1 (APE1), cyclin dependent kinase 3(CDK3), PIM1 kinase (PIM1), cell division cycle 7 kinase (CDC7L1),cyclin dependent kinase 7 (CDK7), cytokine inducible kinase (CNK),potentially prenylated protein tyrosine phosphatase (PRL-3), serinethreonine kinase 2 (STK2) or (NEK4), cyclin dependent serine threoninekinase (NKIAMRE), or histone acetylase (HBO1) polypeptide or fragmentthereof is determined, thereby identifying a compound that modulatescell cycle arrest.

[0008] Yet another embodiment of the invention provides a method ofmodulating cell cycle arrest in a subject. A therapeutically effectiveamount of a compound identified according to one of the methodsdescribed above is administered to the subject. The subject may be ahuman. The subject may have cancer. The compound may inhibit cancer cellproliferation.

[0009] Even another embodiment of the invention provides a method ofmodulating cell cycle arrest in a subject. A therapeutically effectiveamount of a protein kinase C ζ (PKC-ζ), phospholipase C-β1 (PLC-β1),protein tyrosine kinase 2 (FAK), protein tyrosine kinase 2b (FAK2),casein kinase 2 (CK2), cMET tyrosine kinase (cMET), flap structurespecific endonuclease 1 (FEN1), REV1 dCMP transferase (REV1),apurinic/apyrimidinic nuclease 1 (APE1), cyclin dependent kinase 3(CDK3), PIM1 kinase (PIM1), cell division cycle 7 kinase (CDC7L1),cyclin dependent kinase 7 (CDK7), cytokine inducible kinase (CNK),potentially prenylated protein tyrosine phosphatase (PRL-3), serinethreonine kinase 2 (STK2) or (NEK4), cyclin dependent serine threoninekinase (NKIAMRE), or histone acetylase (HBO1) polypeptide isadministered to the subject. The protein kinase C ζ (PKC-ζ),phospholipase C-β1 (PLC-β1), protein tyrosine kinase 2 (FAK), proteintyrosine kinase 2b (FAK2), casein kinase 2 (CK2), cMET tyrosine kinase(cMET), flap structure specific endonuclease 1 (FEN1), REV1 dCMPtransferase (REV1), apurinic/apyrimidinic nuclease 1 (APE1), cyclindependent kinase 3 (CDK3), PIM1 kinase (PIM1), cell division cycle 7kinase (CDC7L1), cyclin dependent kinase 7 (CDK7), cytokine induciblekinase (CNK), potentially prenylated protein tyrosine phosphatase(PRL-3), serine threonine kinase 2 (STK2) or (NEK4), cyclin dependentserine threonine kinase (NKIAMRE), or histone acetylase (HBO1)polypeptide may be encoded by a nucleic acid that hybridizes understringent conditions to a nucleic acid encoding a polypeptide having anamino acid sequence of SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22,24, 26, 28, 30, 32, 34, or 36.

[0010] A further embodiment of the invention provides a method ofmodulating cell cycle arrest in a subject. A therapeutically effectiveamount of anucleic acid encoding a protein kinase C ζ (PKC-ζ),phospholipase C-β1 (PLC-β1), protein tyrosine kinase 2 (FAK), proteintyrosine kinase 2b (FAK2), casein kinase 2 (CK2), cMET tyrosine kinase(cMET), flap structure specific endonuclease 1 (FEN1), REV1 dCMPtransferase (REV1), apurinic/apyrimidinic nuclease 1 (APE1), cyclindependent kinase 3 (CDK3), PIM1 kinase (PIM1), cell division cycle 7kinase (CDC7L1), cyclin dependent kinase 7 (CDK7), cytokine induciblekinase (CNK), potentially prenylated protein tyrosine phosphatase(PRL-3), serine threonine kinase 2 (STK2) or (NEK4), cyclin dependentserine threonine kinase (NKIAMRE), or histone acetylase (HBO1)polypeptide is administered to the subject. The protein kinase C ζ(PKC-ζ), phospholipase C-β1 (PLC-β1), protein tyrosine kinase 2 (FAK),protein tyrosine kinase 2b (FAK2), casein kinase 2 (CK2), cMET tyrosinekinase (cMET), flap structure specific endonuclease 1 (FEN1), REV1 dCMPtransferase (REV1), apurinic/apyrimidinic nuclease 1 (APE1), cyclindependent kinase 3 (CDK3), PIM1 kinase (PIM1), cell division cycle 7kinase (CDC7L1), cyclin dependent kinase 7 (CDK7), cytokine induciblekinase (CNK), potentially prenylated protein tyrosine phosphatase(PRL-3), serine threonine kinase 2 (STK2) or (NEK4), cyclin dependentserine threonine kinase (NKIAMRE), or histone acetylase (HBO1)polypeptide may be encoded by a nucleic acid that hybridizes understringent conditions to a nucleic acid encoding a polypeptide having anamino acid sequence of SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22,24, 26, 28, 30, 32, 34, or 36.

[0011] The invention also provides specific siRNA molecules forinhibition of expression of cell cycle genes. In one embodiment, theinvention provides a CK2-specific siRNA molecule comprising the sequenceAACATTGAATTAGATCCACGT. The CK2-specific siRNA molecule can be from 21 to30 nucleotide base pairs in length. In one aspect, the CK2-specificsiRNA molecule has the sequence AACATTGAATTAGATCCACGT and its complementas active portion. The CK2-specific siRNA molecules can be used in amethod of inhibiting expression of a CK2 gene in a cell, by contactingthe cell with the method comprising contacting the cell with aCK2-specific siRNA molecule from 21 to 30 nucleotide base pairs inlength that includes the sequence AACATTGAATTAGATCCACGT.

[0012] In another embodiment, the invention provides a PIM1-specificsiRNA molecule comprising the sequence AAAACTCCGAGTGAACTGGTC. ThePIM1-specific siRNA molecule can be from 21 to 30 nucleotide base pairsin length. In one aspect, the PIM1-specific siRNA molecule has thesequence AAAACTCCGAGTGAACTGGTC and its complement as active portion. ThePIM1-specific siRNA molecules can be used in a method of inhibitingexpression of a PIM1 gene in a cell, by contacting the cell with themethod comprising contacting the cell with a PIM1-specific siRNAmolecule from 21 to 30 nucleotide base pairs in length that includes thesequence AAAACTCCGAGTGAACTGGTC.

[0013] In another embodiment, the invention provides a Hbo1-specificsiRNA molecule comprising the sequence AACTGAGCAAGTGGTTGATTT. TheHbo1-specific siRNA molecule can be from 21 to 30 nucleotide base pairsin length. In one aspect, the Hbo1-specific siRNA molecule has thesequence AACTGAGCAAGTGGTTGATTT and its complement as active portion. TheHbo1-specific siRNA molecules can be used in a method of inhibitingexpression of a Hbo1 gene in a cell, by contacting the cell with themethod comprising contacting the cell with a Hbo1-specific siRNAmolecule from 21 to 30 nucleotide base pairs in length that includes thesequence AACTGAGCAAGTGGTTGATTT.

[0014] Other embodiments and advantages of the present invention will beapparent from the detailed description that follows.

BRIEF DESCRIPTION OF THE DRAWINGS

[0015]FIG. 1 provides a nucleotide (SEQ ID NO:1) and amino acid (SEQ IDNO:2) sequence of human PKC-ζ.

[0016]FIG. 2 provides a nucleotide (SEQ ID NO:3) and an amino acid (SEQID NO:4) sequence of human PLC-β1.

[0017]FIG. 3 provides a nucleotide (SEQ ID NO:5) and an amino acid (SEQID NO:6) sequence of human FAK.

[0018]FIG. 4 provides a nucleotide (SEQ ID NO:7) and an amino acid (SEQID NO:8) sequence of human FAK2.

[0019]FIG. 5 provides a nucleotide (SEQ ID NO:9) and an amino acid (SEQID NO:10) sequence of human CK2.

[0020]FIG. 6 provides a nucleotide (SEQ ID NO:11) and an amino acid (SEQID NO:12) sequence of human cMET.

[0021]FIG. 7 provides a nucleotide (SEQ ID NO:13) and an amino acid (SEQID NO:14) sequence of human FEN1.

[0022]FIG. 8 provides a nucleotide (SEQ ID NO:15) and an amino acid (SEQID NO:16) sequence of human REV1.

[0023]FIG. 9 provides a nucleotide (SEQ ID NO:17) and an amino acid (SEQID NO:18) sequence of human APE1.

[0024]FIG. 10 provides a nucleotide (SEQ ID NO:19) and an amino acid(SEQ ID NO:20) sequence of human CDK3.

[0025]FIG. 11 provides a nucleotide (SEQ ID NO:21) and an amino acid(SEQ ID NO:22) sequence of human PIM1.

[0026]FIG. 12 provides a nucleotide (SEQ ID NO:23) and an amino acid(SEQ ID NO:24) sequence of human CDC7L1.

[0027]FIG. 13 provides a nucleotide (SEQ ID NO:25) and an amino acid(SEQ ID NO:26) sequence of human CDK7.

[0028]FIG. 14 provides a nucleotide (SEQ ID NO:27) and an amino acid(SEQ ID NO:28) sequence of human CNK.

[0029]FIG. 15 provides a nucleotide (SEQ ID NO:29) and an amino acid(SEQ ID NO:30) sequence of human PRL-3.

[0030]FIG. 16 provides a nucleotide (SEQ ID NO:31) and an amino acid(SEQ ID NO:32) sequence of human STK2 (NEK4).

[0031]FIG. 17 provides a nucleotide (SEQ ID NO:33) and an amino acid(SEQ ID NO:34) sequence of human NKIAMRE.

[0032]FIG. 18 provides a nucleotide (SEQ ID NO:35) and an amino acid(SEQ ID NO:36) sequence of human HBO1.

[0033]FIG. 19 provides a table summarizing genes that may be involved inthe modulation of cell proliferation.

[0034]FIG. 20 illustrates inhibition of proliferation of A549 cells byexpression of wild-type GFP-CDC7LI and mutant GFP-CDC7LI.

[0035]FIG. 21 illustrates inhibition of proliferation of A549 cells byexpression of wild-type CNK and mutant GFP-CNK.

[0036]FIG. 22 illustrates inhibition of proliferation of A549 cells andHela cells by expression of wild-type and mutant STK2.

[0037]FIG. 23 provides amino acid sequences for dominant negativemutants of CDC7L1.

[0038]FIG. 24 provides amino acid sequences for dominant negativemutants of CNK.

[0039]FIG. 25 provides amino acid sequences for dominant negativemutants of STK2.

[0040]FIG. 26 provides an alternative view of the amino acid sequencesfor dominant negative mutants of CDC7L1.

[0041]FIG. 27 provides Taqman analysis (i.e., real time PCR) of Cdc7LmRNA expression using RNA from tumor cell lines and primary human celllines. Cdc7L mRNA levels were normalized to GAPDH mRNA levels.

[0042]FIG. 28 provides analysis of CDC7L mRNA levels in matchedcancerous and normal tissue from patients with lung carcinoma. Eachmatched pair represents a different patient.

[0043]FIG. 29 provides analysis of CDC7L mRNA in matched cancerous andnormal tissue from patients with colon carcinoma. Each matched pairrepresents a different patient.

[0044]FIG. 30 provides Taqman analysis (i.e., real time PCR) of CNK mRNAexpression using RNA from tumor cell lines and primary human cell lines.CNK mRNA levels were normalized to GAPDH mRNA levels.

[0045]FIG. 31 demonstrates that GST-CNK produced in E. coli has kinaseactivity against p53 and MBP. GST-CNK also exhibits autophosphorylationactivity.

[0046]FIG. 32 depicts the structure of STK2 long (STK2L) and short(STK2L) forms and their expression levels in human tissues.

[0047]FIG. 33 provides Taqman analysis (ie., real time PCR) of STK2 mRNAexpression using RNA from tumor cell lines and primary human cell lines.STK2 mRNA levels were normalized to GAPDH mRNA levels.

[0048]FIG. 34 demonstrates that GFP-STK2S expression isantiproliferative when measured using the cell tracker assay.

[0049]FIG. 35 demonstrates that GFP-STK2L expression isantiproliferative in A549 and HeLa cells.

[0050]FIG. 36 demonstrates that GFP-STK2L expression isantiproliferative when measured using the cell tracker assay.

[0051]FIG. 37 demonstrates that IRES-STK2L expression isantiproliferative in A549 and HeLa cells.

[0052]FIG. 38 demonstrates that expression of IRES Hbo1 E508Q isantiproliferative in A549 cells.

[0053]FIG. 39 demonstrates that no significant differences inproliferation are observed between Hbo1 WT and mutant proteins whenexpressed in H 1299 cells.

[0054]FIG. 40 demonstrates that expression of Hbo1 mtant E508Q isantiproliferative in HeLa cells.

[0055]FIG. 41 depicts analysis of proliferation in sorted cells thatexpress wild type or mutant Hbo1 proteins.

[0056]FIG. 42 demonstrates that expression of HBO1 mutant E508Q isantiproliferative in sorted A549 cells.

[0057]FIG. 43 demonstrates that expression of HBO1 mutant E508Q isantiproliferative in sorted HeLa cells.

[0058]FIG. 44 demonstrates that expression of HBO1-specific siRNAreduces Hbo1 mRNA levels and has an antiproliferative effect on A549cells.

[0059]FIG. 45 demonstrates that HBO1-specific siRNA reduces Hbo1 mRNAlevels and has an antiproliferative effect on 1299 cells.

[0060]FIG. 46 provides Taqman analysis (i.e., real time PCR) of PIM1mRNA expression using RNA from tumor cell lines and primary human celllines. PIM1 mRNA levels were normalized to 18S RNA levels.

[0061]FIG. 47 provides Taqman analysis (i.e., real time PCR) of PIM1mRNA levels in matched cancerous and normal tissue from patients withbreast carcinoma. Each matched pair represents a different patient. PIM1mRNA levels were normalized to 18S RNA levels.

[0062]FIG. 48 provides Taqman analysis (i.e., real time PCR) of PIM1mRNA levels in matched cancerous and normal tissue from patients withlung carcinoma. Each matched pair represents a different patient. PIM 1mRNA levels were normalized to 18S RNA levels.

[0063]FIG. 49 demonstrates that expression of PIM1 wild type, but notmutant protein, is antiproliferative in A549 cells.

[0064]FIG. 50 demonstrates that expression of GFP-PIM1 wild type isantiproliferative in H1299 cells. The figure also demonstrates thatexpression of both IRES PIM1 wild type and mutant is antiproliferativein H1299 cells.

[0065]FIG. 51 demonstrates that expression of PIM1-specific siRNAreduces PIM1 mRNA levels and has an antiproliferative effect on A549cells.

[0066]FIG. 52 demonstrates that expression of PIM1-specific siRNAreduces PIM1 mRNA levels and has an antiproliferative effect on HeLacells.

[0067]FIG. 53 demonstrates that expression of PIM1-specific siRNAreduces PIM1 mRNA levels and has an antiproliferative effect on H1299cells.

[0068]FIG. 54 demonstrates that expression of PIM1-specific siRNAreduces PIM1 mRNA levels and has an antiproliferative effect on primaryHUVEC cells.

[0069]FIG. 55 demonstrates that expression of APE1 wild type and mutantproteins is not antiproliferative in A549 cells.

[0070]FIG. 56 demonstrates that expression of APE1 wild type and mutantproteins is not antiproliferative in H1299 cells.

[0071]FIG. 57 demonstrates that expression of APE1 wild type and APE1D210A mutant proteins is antiproliferative in primary HMEC cells.

[0072]FIG. 58 demonstrates that expression of the Ape1 D210A mutantsensitizes A549 cells to methyl methanesulfonate treatment.

[0073]FIG. 59 demonstrates that wild type Ape1 and the Ape1 C65A mutantare protective when expressed in A549 cells treated with bleomycin.

[0074]FIG. 60 demonstrates that wild type Ape1 and the Ape1 C65A mutantare protective when expressed in HeLa cells or H1299 cells treated withbleomycin.

[0075]FIG. 61 provides Taqman analysis (i.e., real time PCR) of CK2αmRNA expression using RNA from tumor cell lines and primary cell lines.CK2α mRNA levels were normalized to 18S RNA levels.

[0076]FIG. 62 provides the sequence of dominant negative mutants ofCK2α.

[0077]FIG. 63 demonstrates that expression of CK2α-specific siRNAreduces CK2α mRNA levels and has an antiproliferative effect on H1299cells.

[0078]FIG. 64 provides Taqman analysis (i.e., real time PCR) of NKIAMREexpression using RNA from tumor cell lines and primary cell lines.NKIAMRE mRNA levels were normalized to 18S RNA levels.

[0079]FIG. 65 provides the sequence of dominant negative mutants ofNKIAMRE.

[0080]FIG. 66 provides the sequence of dominant negative mutants ofFEN1.

[0081]FIG. 67 demonstrates that expression of FEN1 dominant negativemutants in A549 cells is antiproliferative.

[0082]FIG. 68 demonstrates that expression of FEN1 dominant negativemutants in H1299 cells is antiproliferative.

[0083]FIG. 69 provides the sequence of dominant negative mutants ofCDK3.

[0084]FIG. 70 demonstrates that expression of GFP-CDK3 wild type andCDK3 mutant proteins appears to have no antiproliferative effect in A549cells. The figure also demonstrates that expression of both IRES CDK3wild type and CDK3 mutant proteins appears to have no antiproliferativeeffect in A549 cells.

[0085]FIG. 71 demonstrates that expression of GFP-CDK3 wild type andCDK3 mutant proteins appears to have no antiproliferative effect inH1299 cells. The figure also demonstrates that expression of both IRESCDK3 wild type and CDK3 mutant proteins appears to have noantiproliferative effect in H1299 cells.

[0086]FIG. 72 provides the sequence of dominant negative mutants ofHBO1.

[0087]FIG. 73 provides the sequence of dominant negative mutants ofPIM1.

[0088]FIG. 74 demonstrates that expression of GFP-NKIAMRE wild type andNKIAMRE mutant proteins appears to have no antiproliferative effect ineither A549 cells or H1299 cells.

DETAILED DESCRIPTION OF THE INVENTION

[0089] Introduction

[0090] PKC-ζ, PLC-β1, FAK, FAK2, CK2, cMET, FEN1, REV1, APE1, CDK3,PIM1, CDC7L1, CDK7, CNK, PRL-3, STK2 (NEK4), NKIAMRE, and HBO1 encodeproteins involved in modulation of the cell cycle in cancer cells.

[0091] As described below, the present inventors identified PKC-ζ,PLC-β1, FAK, FAK2, CK2, cMET, FEN1, REV1, APE1, CDK3, PIM1, CDC7L1,CDK7, CNK, PRL-3, STK2 (NEK4), NKIAMRE, and HBO1 as modulators of thecell cycle in immunoprecipitation assays or yeast 2 hybrid assays.

[0092] PKC-ζ encodes an a typical isoform of protein kinase C, i.e., anisoform that is not activated by phorbol esters or diacylglycerols (see,e.g., Donson et al. J. Neuro-Onc., 47:109 (2000)). PKC-ζ activatesseveral signaling pathways, mediates multiple cellular functions, andplays a role in the proliferation of fibroblast cells, endothelialcells, smooth muscle cells, human glioblastoma cells, and astrocytomacells (see, e.g., Guizzetti and Costa, Biochem. Pharmacol., 60:1457(2000); Donson et al., 2000). PKC-ζ also plays a role in the activationof p70 S6 kinase which modulates the progression through the G₁ phase ofthe cell cycle (see, Guizzetti, 2000). Assays known to those of skill inthe art can be used to identify modulators of PKC-ζ (see, e.g., J. Biol.Chem., 276:3543; J. Biol. Chem., 272:31130; J. Biol. Chem., 270:15884;J. Biol. Chem., 273:26277; J. Biol. Chem., 272:16578; Mol. Cell. Biol.,19:2180). For example, IRS-1, nucleoli, heterogeneous ribonucleoproteinA1, Sp1, Sendai virus phosphoprotein, and IKK β may be used assubstrates in assays to identify modulators of PKCζ (see, e.g., J. Biol.Chem., 276:3543; J. Biol. Chem., 272:31130; J. Biol. Chem., 270:15884;J. Biol. Chem., 273:26277; J. Biol. Chem., 272:16578; Mol. Cell. Biol.,19:2180).

[0093] PLC-β1 encodes a phosphoinositide-specific phospholipase C. ThePLC-β1 isoform is the predominant nuclear phospholipase C in multiplecell types, including erythroleukemia cells, osteosarcoma cells,pheochromocytoma cells, and glioma cells (see, e.g., Cocco et al.,Advan. Enzyme Regul., 39:287 (1999)). PLC-β1 has been shown tobe-responsible for nuclear inositol lipid metabolism in multiple celltypes (see, e.g., Avazeri, et al., Mol. Biol. Cell, 11:4369 (2000)).Overexpression of PLC-β1 in human colon cancer cells suppresses tumorcell growth, but induces increased cell aggregation and increasedexpression and release of carcinoembryonic antigen molecule (see, e.g.,Nomoto et al., Jpn. J. Canc. Res., 89:1257 (1998)). PLC-β1 has beenreported to be essential for IGF-1 induced mitogenesis (see, Cocco etal., 1999). Phospholipase C activity assays known to those of skill inthe art can be used to identify modulators of PLC-β1 (see, e.g., Nomotoet al., 1998; Physiol. Rev., 80:1291 (2000); Biochemistry, 36:848; Eur.J. Biochem., 213:339). For example, phosphoinositide may be used as asubstrate in assays to identify modulators of PLC-β1 (see, e.g., Nomotoet al., 1998; and Physiol. Rev., 80:1291 (2000); Biochemistry, 36:848;Eur. J. Biochem., 213:339). Additional assays to identify modulators ofPLC-β1 are described in, e.g., 109 Mark Dolittle and Karen Reue, Methodsin Molecular Biology: Lipase and Phospholipase Protocols (1998)

[0094] FAK encodes a cytoplasmic tyrosine kinase that plays a role inregulation of cell cycle progression (see, e.g., MacPhee et al., Lab.Invest., 81(11):1469 (2001) and Zhao et al., Mol. Biol. Cell, 12:4066(2001)). Specifically, FAK regulates cell cycle progression byincreasing cyclin D1 expression and/or decreasing expression of the CDKinhibitor p21 (see, Zhao et al., 2001). High levels of FAK have beenlinked to tumor invasiveness and metastasis (see, e.g., Fresu et al.,Biochem. J., 358:407 (2001)). Tyrosine kinase assays known to those ofskill in the art can be used to identify modulators of FAK (see, e.g.,Bioessays, 19:137; Mol. Biol. Cell, 10:2507 (1999)). For example,p130Cas and paxillin may be used as a substrate to identify modulatorsof FAK (see, e.g., Bioessays, 19:137; Mol. Biol. Cell, 10:2507 (1999)).

[0095] FAK2 encodes a calcium dependent tyrosine kinase that localizesto sites of cell-to-cell contact and participates in cellular signaltransduction (see, e.g., Sasaki et al., J. Bio. Chem., 270(6):21206(1995) and Li et al., J. Biol. Chem., 273(16):9361 (1998)). Tyrosinekinase assays known to those of skill in the art can be used to identifymodulators of FAK2 (see, e.g., Sasaki et al., 1995). For example,p130Cas and paxillin may be used as substrates in assays to identifymodulators of FAK2.

[0096] CK2 or CK2α encodes an ubiquitous serine threonine protein kinasethat is required for the G₂/M transition and checkpoint control stagesof the cell cycle (see, e.g. Messenger et al., J. Biol. Chem. 277:23054(2002), Sayed et al., Oncogene 20(48):6994 (2001), and Escargueil et al.J. Biol. Chem. 275(44):34710 (2000)). In particular, CK2 is required forthe phosphorylation of topoisomerase 1 during the G₂/M transition of thecell cycle (see, Messenger et al., 2002). CK2 is overexpressed in tumorsand leukemic cells (see, Messenger et al., 2002). CK2 works with p53 inspindle checkpoint arrest to maintain increase cyclin B/cdc2 kinaseactivity (see, Sayed et al., 2001). Serine threonine protein kinaseassays known to those of skill in the art can be used in assays toidentify modulators of CK2 (see, e.g., Messenger et al., 2002 and J.Biol. Chem., 274(41):29260).

[0097] cMET encodes a tyrosine kinase that is expressed in numeroustissues and plays a role in the generation and spread of tumors of thestomach, rectum, lung, pancreas, breast, and bile duct (see, e.g.,Jeffers et al., Proc. Nat'l. Acad. Sci. USA 94:11445 (1997) and Ramirezet al., Endocrinology 53:635 (2000)). More specifically, cMET plays arole in angiogenesis, cell motility, cell growth, cell invasion, andmorphogenic differentiation (see, Jeffers et al., 1997). In particular,cMET overexpression is associated with a high risk of metastasis andrecurrence of papillary thyroid carcinoma (see, Ramirez et al., 2000).Tyrosine kinase assays known to those of skill in the art can be used inassays to identify modulators of cMET (see, Jeffers et al., 1997). Forexample dCMP, Grb2, Gab can be used as substrates in assays to identifymodulators of cMET.

[0098] FEN1 encodes a structure specific endonuclease that cleavessubstrates with unannealed 5′ tails (see, e.g., Warbrick et al., J.Pathol. 186:319 (1998)). FEN1 has high specificity of binding/activitytoward 5′ flap structures, i.e., dsDNA with a displaced 5′ strand (see,e.g., Warbrick et al., 1998 and Tom et al., J. Biol. Chem. 275(14):10498(2000)). FEN1 also exhibits a 5′ to 3′ exonucleolytic activity. FEN1levels are low in non-cycling cells and are induced as the cells enterthe cell cycle (see, Warbrick et al., 1998). FEN1 assays known to thoseof skill in the art can be used to identify modulators of FEN1 (see, Tomet al., 2000 and EMBO J., 13(5):1235 (1994)). For example, 5′ DNA flapstructures can be used as substrates in assays to identify modulators ofFEN1 (see, e.g., EMBO J., 13(5):1235 (1994)).

[0099] REV1 encodes a 1251 amino acid dCMP transferase that functions inthe Polζ mutagenesis pathway (see, e.g., Lui et al., Nuc. Acids. Res.27(22):4468 (1999) and Zhang et al., Nuc. Acids Res. 30(7):1630 (2002)).REV1 has been implicated in UV induced mutagenesis repair and ispostulated to play a role in UV damage tolerance (see, e.g., Murakomo,J. Biol. Chem., 276(38):35644 (2001)). dCMP transferase assays known tothose of skill in the art can be used to identify modulators of REV1(see, Zhang et al., 2002 and J. Biol. Chem., 276(18):15051). Forexample, dCMP, 5′-end 32P-labeled oligonucleotide primer5′-CACTGACTGTATG-3′ annealed to an oligonucleotide template,5′-CTCGTCAGCATCTTCAUCATACAGTCAGTG-3′ treated with uracil-DNA glycosylasemay be used as substrates in assays to identify modulators of REV1 (see,e.g., J. Biol. Chem., 276(18):15051).

[0100] APE1 encodes an apyrimidinic endonuclease that plays a role inshort patch repair and long patch repair of ionizing radiation andalkyklating agent induced damage in DNA (see, e.g., Tom et al., J. Biol.Chem., 276(52):48781 (2001), Izumi, Carcinogenesis, 21(7):1329 (2000),and Bobola et al., Clin. Cancer Res. 7(11):3510 (2001)). APE1 has alsoplays a role the cellular response to oxidative stress, regulation oftranscription factors, cell cycle control, and apoptosis (see, Bobola etal., 2001). Assays known to those of skill in the art can be used toidentify modulators of APE1 (see, Tom et al., 2001 and Bobola et al.,2001; Nucleic Acids Res., 5(4):1413 (1978); Biochimie, 64(8-9):603(1982); Mutat. Res., 460(3-4):211 (2000)). For example, oligonucleotideduplexes containing an apurinic/apyrimidinic sites may be used as asubstrate in assays to identify modulators of APE1.

[0101] CDK3 encodes a cyclin dependent kinase that regulates entry intoS phase. (see, e.g., Braun et al., Oncogene, 17(7):2259 (1998)).Specifically, CDK3 has been described as a positive G₁ phase regulatorthat enhances the G₁/S transition (see, Braun et al., Oncogene, 1998).Overexpression of CDK2 and CDK3 together has been show to elevate c-mycinduced apoptosis (see, e.g., Braun et al., DNA Cell Biol., 17(9):789(1998)). A dominant negative mutant of CDK3 suppresses apoptosis andoverexpression of CDK3 circumvents the anti-apoptotic effect of bcl-2(see, e.g., Meikrantz and Schlegel, J. Biol. Chem., 271(17):10205(1996)). Assays known to those of skill in the art can be used toidentify modulators of CDK3 (see, e.g., Eur. J. Biochem., 268:6076(2001)). For example, pRb, histone H1, and P701K3-1 (the C-terminaldomain of RNA Pol I) may used as substrates in assays to identifymodulators of CDK3 (see, e.g., Eur. J. Biochem., 268:6076 (2001)).

[0102] PIM1 encodes two cytoplasmic serine threonine kinases generatedby an alternate translation initiation (see, e.g., Mochizuki et al.,Oncogene 15:1471 (1997) and Shirogane et al., Immunity 11:709 (1999)).PIM1 plays a role in cellular transformation and inhibits apoptosis(see, e.g., Mochizuki et al., 1997). Specifically, PIM1 cooperates withc-myc to promote cell proliferation through the G₁ to S transition andto prevent apoptosis (Shirogane et al., 1999). PIM1 has been implicatedin T cell lymphoma, i.e., it has been shown that PIM1 cooperates withthe oncoprotein E2α-Pbx1 to facilitate thymic lymphagenesis (see, e.g.,Feldman et al., Oncogene 15(22):2735 (1997)). Assays known to those ofskill in the art can be used to identify modulators of PIM1 (see, e.g.,J. Biol. Chem., 266(21):14018). For example, histone H1 may be used as asubstrate in assays to identify modulators of PIM1 (see, e.g., J. Biol.Chem., 266 (21):14018).

[0103] CDC7L1 encodes a 574 amino acid serine threonine kinase (see,e.g., Masai and Arai, J. Cell Physiol., 190(3):287 (2002), Masai et al.,J. Biol. Chem., 275(37):29042 (2000), and Johnston et al., Prog. CellCycle Res., 4:61(2002)). CDC7L1 binds the activator for S phase kinase(ASK) to form a complex that is present at high levels during S phaseand decreased levels during G₁ phase. Assays known to those of skill inthe art can be used to identify modulators of CDC7L1 (see, e.g., Masaiet al., 2000; Johnston et al., 2000; and Proc. Natl. Acad. Sci. USA,94:14320 (1997)). For example, histone H1 may be used as a substrate inassays to identify modulators of CDC7L1 (see, e.g., Proc. Natl. Acad.Sci. USA, 94:14320 (1997)). Alternatively, Mcm2 may be used as asubstrate in assays to identify modulators of CDC7L1 (see, e.g., Takedaet al., Mol. Biol. Cell, 12:1257 (2001)). Conditional muCDC7-deficientembryonic cell lines and transgenic CDC7 knockout mice have beengenerated (see, e.g., EMBO J. 21L2168 (2002). The cell lines undergo Sphase arrest and the knockout mouse is embryonic lethal.

[0104] CDK7 encodes a cyclin dependent kinase that is postulated to playa role in cell cycle regulation (see, e.g., Nishiwaki et al., Mol. CellBiol., 20(20):7726 (2000), Acevedo-Duncan et al., Cell. Prolif. 35(1):23(2002), and Bregman et al., Front. Biosci., 5:D244 (2000)). CDK7 is thekinase component of the transcription factor complex TFIIH and has beenshown to contribute to the ability of p16^(INK4A) to induce cell cyclearrest (see, Nishiwaki et al., 2002). Assays known to those of skill inthe art can be used to identify modulators of CDK7 (see, e.g., Mol.Cell. Biol., 21:88 (2001)). For example, CDK2 and the C-terminal domainof RNA Pol II can be used as substrates in assays to identify modulatorsof CDK7.

[0105] CNK is also known as PRK (Proliferation related kinase) andencodes a cytokine inducible serine threonine kinase (see, e.g., Li etal., J. Biol. Chem. 271 (32):19402 (1996), Dai et al., Genes ChromosomesCancer, 27(3):332 (2000), and Ouyang et al., Oncogene, 18(44):6029(1999)). CNK is a member of the polo family of kinases which have beenimplicated in cell division (see, Li et al., 1996). CNK expression isdownregulated in lung cancer and in head and neck cancer (see, Li etal., 1996 and Dai et al., 2000). Assays known to those of skill in theart can be used to identify modulators of CNK (see, e.g., J. Biol.Chem., 272:28646). For example, CDC25, p53, and casein can be used assubstrates in assays to identify modulators of CNK (see, e.g., J. Biol.Chem., 272:28646).

[0106] PRL-3 encodes a 22 kDa potentially prenylated protein tyrosinephosphatase (see, e.g., Zeng et al., Biochem. Biophys. Res. Commun.244(2):421 (1998), Saha et al., Science, 294(5545):1343 (2001), andBradbury, Lancet 358(9289):1245 (2001)). PRL-3 is localized to thecytoplasmic membrane when prenylated at its carboxy terminus, and to thenucleus when it is not prenylated (see, Saha et al., 2001). PRL-3 isexpressed at low levels in normal colorectal epithelial cells, atintermediate levels in malignant stage I or II cancers, and at highlevels in colorectal metastases (see, Saha et al., 2001). Assays knownto those of skill in the art can be used to identify modulators ofPRL-3.

[0107] STK2 is also known as NEK4 and encodes a serine threonine kinase(see, e.g., Chen et al., Gene, 234(1):127 (1999), Hayashi et al.,Biochem. Biophys. Res. Commun., 264(2):449 (1999) and Levedakou et al.,Oncogene 9(7):1977 (1994). STK2 (NEK4) has been localized to chromosome3p21.1 and is a member of the NIMA family of kinases which are G₂/Mregulators of the cell cycle. Assays known to those of skill in the artcan be used to identify modulators of STK2 (NEK4) (see, Hayashi et al.,1999; Biochem. Biophys. Res. Commun. 264(2):449 (1999); J. Biol. Chem.269:6603 (1994)). For example, the polypeptide FRXT can be used as asubstrate in assays to modulate STK2 function.

[0108] NKIAMRE encodes the human homologue to the mitogen-activatedprotein kinase-/cyclin-dependent kinase-related protein kinase NKIATRE(see, e.g., Midermer et al., Cancer Res., 59(16):4069 (1999)). NKIAMRElocalizes to chromosome band 5q31 and is deleted in samples fromleukemia patients (see, e.g., Midermer et al., 1999). Assays known tothose of skill in the art can be used to identify modulators of NKIAMRE.

[0109] HBO1 encodes a member of the MYST family of histoneacetyltransferases (see, e.g., Iizuka and Stillman, J. Biol. Chem.,274(33):23027 (1999), Sterner and Berger, Microbiol. Mol. Biol. Rev.,64(2):435 (2000), and Burke et al., J. Biol. Chem. 276(18):15397(2001)). HBO1 binds to ORC (origin recognition complex) to form acomplex that plays a role in the initiation of replication (see, Sternerand Berger, 2000). Assays known to those of skill in the art can be usedto identify modulators of HBO1 (see, Iizuka and Stillman, 1999 and J.Bio. Chem., 274 (33):23027 (1999)). For example, histone H3 and histoneH4 can be used as substrates in assays to identify modulators of HBO1(see, e.g., J. Bio. Chem., 274(33):23027 (1999)).

[0110] Thus, protein kinase C ζ (PKC-ζ), phospholipase C-β1 (PLC-β1),protein tyrosine kinase 2 (FAK), protein tyrosine kinase 2b (FAK2),casein kinase 2 (CK2), cMET tyrosine kinase (cMET), flap structurespecific endonuclease 1 (FEN1), REV1 dCMP transferase (REV1),apurinic/apyrimidinic nuclease 1 (APE1), cyclin dependent kinase 3(CDK3), PIM1 kinase (PIM1), cell division cycle 7 kinase (CDC7L1),cyclin dependent kinase 7 (CDK7), cytokine inducible kinase (CNK),potentially prenylated protein tyrosine phosphatase (PRL-3), serinethreonine kinase 2 (STK2) or (NEK4), cyclin dependent serine threoninekinase (NKIAMRE), and histone acetylase (HBO1) can conveniently be usedto identify agents that modulate the cell cycle.

[0111] PKC-ζ, PLC-β1, FAK, FAK2, CK2, cMET, FEN1, REV1, APE1, CDK3,PIM1, CDC7L1, CDK7, CNK, PRL-3, STK2 (NEK4), NKIAMRE, and HBO1 thereforerepresent drug targets for compounds that suppress or activate cellularproliferation in tumor cells, or cause cell cycle arrest, cause releasefrom cell cycle arrest, activate apoptosis, increase sensitivity tochemotherapeutic (adjuvant) reagents, and decrease toxicity ofchemotherapeutic reagents. Agents identified in these assays, includingsmall organic molecules, peptides, cyclic peptides, nucleic acids,antibodies, antisense nucleic acids, RNAi, and ribozymes, that modulatecell cycle regulation and cellular proliferation via modulation ofPKC-ζ, PLC-β1, FAK, FAK2, CK2, cMET, FEN1, REV1, APE1, CDK3, PIM1,CDC7L1, CDK7, CNK, PRL-3, STK2 (NEK4), NKIAMRE, or HBO1, can be used totreat diseases related to cellular proliferation, such as cancer. Inparticular, inhibitors of PKC-ζ, PLC-β1, FAK, FAK2, CK2, cMET, FEN1,REV1, APE1, CDK3, PIM1, CDC7L1, CDK7, CNK, PRL-3, STK2 (NEK4), NKIAMRE,or HBO1 are useful for inhibition of cancer and tumor cell growth.PKC-ζ, PLC-β1, FAK, FAK2, CK2, cMET, FEN1, REV1, APE1, CDK3, PIM1,CDC7L1, CDK7, CNK, PRL-3, STK2 (NEK4), NKIAMRE, or HBO1 modulators canalso be used to modulate the sensitivity of cells to chemotherapeuticagents, such as bleomycin, etoposide, taxol, and other agents known tothose of skill in the art. PKC-ζ, PLC-β1, FAK, FAK2, CK2, cMET, FEN1,REV1, APE1, CDK3, PIM1, CDC7L1, CDK7, CNK, PRL-3, STK2 (NEK4), NKIAMRE,or HBO1 modulators can also be used to decrease toxicity of suchchemotherapeutic reagents.

[0112] In one embodiment, enzymatic assays, including kinase orautophosphorylation assays, lipase assays, nuclease assays, transferaseassays, phosphatase assays, and acetylase assays using PKC-ζ, PLC-β1,FAK, FAK2, CK2, cMET, FEN1, REV1, APE1, CDK3, PIM1, CDC7L1, CDK7, CNK,PRL-3, STK2 (NEK4), NKIAMRE, or HBO1 can be used to identify modulatorsof PKC-ζ, PLC-β1, FAK, FAK2, CK2, cMET, FEN1, REV1, APE1, CDK3, PIM1,CDC7L1, CDK7, CNK, PRL-3, STK2 (NEK4), NKIAMRE, or HBO1 activity, or toidentify proteins that bind to PKC-ζ, PLC-β1, FAK, FAK2, CK2, cMET,FEN1, REV1, APE1, CDK3, PIM1, CDC7L1, CDK7, CNK, PRL-3, STK2 (NEK4),NKIAMRE, or HBO1, e.g., PKC-ζ, PLC-β1, FAK, FAK2, CK2, cMET, FEN1, REV1,APE1, CDK3, PIM1, CDC7L1, CDK7, CNK, PRL-3, STK2 (NEK4), NKIAMRE, orHBO1 substrates. Full length wild type PKC-ζ, PLC-β1, FAK, FAK2, CK2,cMET, FEN1, REV1, APE1, CDK3, PIM1, CDC7L1, CDK7, CNK, PRL-3, STK2(NEK4), NKIAMRE, or HBO1, mutant PKC-ζ, PLC-β1, FAK, FAK2, CK2, cMET,FEN1, REV1, APE1, CDK3, PIM1, CDC7L1, CDK7, CNK, PRL-3, STK2 (NEK4),NKIAMRE, or HBO1 can be used in these assays.

[0113] Such modulators are useful for treating cancers, such asmelanoma, breast, ovarian, lung, gastrointestinal and colon, prostate,and leukemia and lymphomas, e.g., multiple myeloma. In addition, suchmodulators are useful for treating noncancerous disease states caused bypathologically proliferating cells such as thyroid hyperplasia (Grave'sdisease), psoriasis, benign prostatic hypertrophy, neurofibromas,atherosclerosis, restenosis, and other vasoproliferative disease.

[0114] Definitions

[0115] By “disorder associated with cellular proliferation” or “diseaseassociated with cellular proliferation” herein is meant a disease statewhich is marked by either an excess or a deficit of cellularproliferation or apoptosis. Such disorders associated with increasedcellular proliferation include, but are not limited to, cancer andnon-cancerous pathological proliferation.

[0116] The terms “PKC-ζ, PLC-β1, FAK, FAK2, CK2, cMET, FEN1, REV1, APE1,CDK3, PIM1, CDC7L1, CDK7, CNK, PRL-3, STK2 (NEK4), NKIAMRE, or HBO1” ora nucleic acid encoding “PKC-ζ, PLC-β1, FAK, FAK2, CK2, cMET, FEN1,REV1, APE1, CDK3, PIM1, CDC7L1, CDK7, CNK, PRL-3, STK2 (NEK4), NKIAMRE,or HBO1” refer to nucleic acids and polypeptide polymorphic variants,alleles, mutants, and interspecies homologs that: (1) have an amino acidsequence that has greater than about 60% amino acid sequence identity,65%, 70%, 75%, 80%, 85%, 90%, preferably 91%, 92%, 93%, 94%, 95%, 96%,97%, 98% or 99% or greater amino acid sequence identity, preferably overa region of over a region of at least about 25, 50, 100, 200, 500, 1000,or more amino acids, to an amino acid sequence encoded by a PKC-ζ,PLC-β1, FAK, FAK2, CK2, cMET, FEN1, REV1, APE1, CDK3, PIM1, CDC7L1,CDK7, CNK, PRL-3, STK2 (NEK4), NKIAMRE, or HBO1 nucleic acid (for ahuman PKC-ζ, PLC-β1, FAK, FAK2, CK2, cMET, FEN1, REV1, APE1, CDK3, PIM1,CDC7L1, CDK7, CNK, PRL-3, STK2 (NEK4), NKIAMRE, or HBO1 nucleic acidsequence, see, e.g., FIGS. 1-18, SEQ ID NO:1, 3, 5, 7, 9, 11, 13, 15,17, 19, 21, 23, 25, 27, 29, 31, 33, 35 or Accession number NM_(—)002744,NM_(—)015192, L05186, L49207, NM_(—)001895, J02958, NM_(—)004111,AF206019, X66133, NM_(—)001258, M16750, NM_(—)003503, NM_(—)001799,NM_(—)004073, NM_(—)007079, XM_(—)003216, AF130372, or NM_(—)007067 oramino acid sequence of a PKC-ζ, PLC-β1, FAK, FAK2, CK2, cMET, FEN1,REV1, APE1, CDK3, PIM1, CDC7L1, CDK7, CNK, PRL-3, STK2 (NEK4), NKIAMRE,or HBO1 protein (for a human PKC-ζ, PLC-β1, FAK, FAK2, CK2, cMET, FEN1,REV1, APE1, CDK3, PIM1, CDC7L1, CDK7, CNK, PRL-3, STK2 (NEK4), NKIAMRE,or HBO1 protein sequence, see, e.g., FIGS. 1-18, SEQ ID NO:2, 4, 6, 8,10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36 or Accessionnumber AAA36488, NP_(—)056007, AAA35819, Q14289, NP_(—)001886, AAA59591,NP_(—)004102, AAF18986, S34422, NP_(—)001249, AAA60089, NP_(—)003494,NP_(—)001790, NP_(—)004064, NP_(—)009010, XP_(—)003216, AAF36509, andNP_(—)008998; (2) bind to antibodies, e.g., polyclonal antibodies,raised against an immunogen comprising an amino acid sequence of aPKC-ζ, PLC-β1, FAK, FAK2, CK2, cMET, FEN1, REV1, APE1, CDK3, PIM1,CDC7L1, CDK7, CNK, PRL-3, STK2 (NEK4), NKIAMRE, or HBO1 protein, andconservatively modified variants thereof; (3) specifically hybridizeunder stringent hybridization conditions to an anti-sense strandcorresponding to a nucleic acid sequence encoding a PKC-ζ, PLC-β1, FAK,FAK2, CK2, cMET, FEN1, REV1, APE1, CDK3, PIM1, CDC7L1, CDK7, CNK, PRL-3,STK2 (NEK4), NKIAMRE, or HBO1 protein, and conservatively modifiedvariants thereof; (4) have a nucleic acid sequence that has greater thanabout 95%, preferably greater than about 96%, 97%, 98%, 99%, or highernucleotide sequence identity, preferably over a region of at least about25, 50, 100, 200, 500, 1000, or more nucleotides, to a PKC-ζ, PLC-β1,FAK, FAK2, CK2, cMET, FEN1, REV1, APE1, CDK3, PIM1, CDC7L1, CDK7, CNK,PRL-3, STK2 (NEK4), NKIAMRE, or HBO1 nucleic acid or a nucleic acidencoding the enzymatic domain. Preferably the enzymatic domain hasgreater than 96%, 97%, 98%, or 99% amino acid identity to the humanPKC-ζ, PLC-β1, FAK, FAK2, CK2, cMET, FEN1, REV1, APE1, CDK3, PIM1,CDC7L1, CDK7, CNK, PRL-3, STK2 (NEK4), NKIAMRE, or HBO1 enzymatic domainof SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32,35, or 36. A polynucleotide or polypeptide sequence is typically from amammal including, but not limited to, primate, e.g., human; rodent,e.g., rat, mouse, hamster; cow, pig, horse, sheep, or any mammal. Thenucleic acids and proteins of the invention include both naturallyoccurring or recombinant molecules.

[0117] The phrase “functional effects” in the context of assays fortesting compounds that modulate activity of a PKC-ζ, PLC-β1, FAK, FAK2,CK2, cMET, FEN1, REV1, APE1, CDK3, PIM1, CDC7L1, CDK7, CNK, PRL-3, STK2(NEK4), NKIAMRE, or HBO1 protein includes the determination of aparameter that is indirectly or directly under the influence of a PKC-ζ,PLC-β1, FAK, FAK2, CK2, cMET, FEN1, REV1, APE1, CDK3, PIM1, CDC7L1,CDK7, CNK, PRL-3, STK2 (NEK4), NKIAMRE, or HBO1, e.g., a phenotypic orchemical effect, such as the ability to increase or decrease cellularproliferation, apoptosis, cell cycle arrest, or enzymatic activity, ore.g., a physical effect such as ligand binding or inhibition of ligandbinding. A functional effect therefore includes ligand binding activity,the ability of cells to proliferate, apoptosis, and enzyme activity.“Functional effects” include in vitro, in vivo, and ex vivo activities.

[0118] By “determining the functional effect” is meant assaying for acompound that increases or decreases a parameter that is indirectly ordirectly under the influence of a PKC-ζ, PLC-β1, FAK, FAK2, CK2, cMET,FEN1, REV1, APE1, CDK3, PIM1, CDC7L1, CDK7, CNK, PRL-3, STK2 (NEK4),NKIAMRE, or HBO1 protein, e.g., measuring physical and chemical orphenotypic effects. Such functional effects can be measured by any meansknown to those skilled in the art, e.g., changes in spectroscopiccharacteristics (e.g., fluorescence, absorbance, refractive index);hydrodynamic (e.g., shape); chromatographic; or solubility propertiesfor the protein; measuring inducible markers or transcriptionalactivation of the protein; measuring binding activity or binding assays,e.g. binding to antibodies; measuring changes in ligand or substratebinding activity; measuring cellular proliferation; measuring cellmorphology, e.g., spindle formation or chromosome formation; measuringphosphorylated proteins such as histone H3 using antibodies; measuringapoptosis; measuring cell surface marker expression; measurement ofchanges in protein levels for PKC-ζ, PLC-β1, FAK, FAK2, CK2, cMET, FEN1,REV1, APE1, CDK3, PIM1, CDC7L1, CDK7, CNK, PRL-3, STK2 (NEK4), NKIAMRE,or HBO1-associated sequences; measurement of RNA stability,identification of downstream or reporter gene expression (CAT,luciferase, β-gal, GFP and the like), e.g., via chemiluminescence,fluorescence, colorimetric reactions, antibody binding, and induciblemarkers.

[0119] “Inhibitors”, “activators”, and “modulators” of PKC-ζ, PLC-β1,FAK, FAK2, CK2, cMET, FEN1, REV1, APE1, CDK3, PIM1, CDC7L1, CDK7, CNK,PRL-3, STK2 (NEK4), NKIAMRE, or HBO1 polynucleotide and polypeptidesequences are used to refer to activating, inhibitory, or modulatingmolecules identified using in vitro and in vivo assays of PKC-ζ, PLC-β1,FAK, FAK2, CK2, cMET, FEN1, REV1, APE1, CDK3, PIM1, CDC7L1, CDK7, CNK,PRL-3, STK2 (NEK4), NKIAMRE, or HBO1 polynucleotide and polypeptidesequences. Inhibitors are compounds that, e.g., bind to, partially ortotally block activity, decrease, prevent, delay activation, inactivate,desensitize, or down regulate the activity or expression of PKC-ζ,PLC-β1, FAK, FAK2, CK2, cMET, FEN1, REV1, APE1, CDK3, PIM1, CDC7L1,CDK7, CNK, PRL-3, STK2 (NEK4), NKIAMRE, or HBO1 proteins, e.g.,antagonists. “Activators” are compounds that increase, open, activate,facilitate, enhance activation, sensitize, agonize, or up regulatePKC-ζ, PLC-β1, FAK, FAK2, CK2, cMET, FEN1, REV1, APE1, CDK3, PIM1,CDC7L1, CDK7, CNK, PRL-3, STK2 (NEK4), NKIAMRE, or HBO1 proteinactivity, e.g., agonists. Inhibitors, activators, or modulators alsoinclude genetically modified versions of PKC-ζ, PLC-β1, FAK, FAK2, CK2,cMET, FEN1, REV1, APE1, CDK3, PIM1, CDC7L1, CDK7, CNK, PRL-3, STK2(NEK4), NKIAMRE, or HBO1 proteins, e.g., versions with altered activity,as well as naturally occurring and synthetic ligands, antagonists,agonists, antibodies, peptides, cyclic peptides, nucleic acids, siRNAmolecules, antisense molecules, ribozymes, small chemical molecules andthe like. Such assays for inhibitors and activators include, e.g.,expressing PKC-ζ, PLC-β1, FAK, FAK2, CK2, cMET, FEN1, REV1, APE1, CDK3,PIM1, CDC7L1, CDK7, CNK, PRL-3, STK2 (NEK4), NKIAMRE, or HBO1 protein invitro, in cells, or cell membranes, applying putative modulatorcompounds, and then determining the functional effects on activity, asdescribed above.

[0120] Samples or assays comprising PKC-ζ, PLC-β1, FAK, FAK2, CK2, cMET,FEN1, REV1, APE1, CDK3, PIM1, CDC7L1, CDK7, CNK, PRL-3, STK2 (NEK4),NKIAMRE, or HBO1 proteins that are treated with a potential activator,inhibitor, or modulator are compared to control samples without theinhibitor, activator, or modulator to examine the extent of inhibition.Control samples (untreated with inhibitors) are assigned a relativeprotein activity value of 100%. Inhibition of PKC-ζ, PLC-β1, FAK, FAK2,CK2, cMET, FEN1, REV1, APE1, CDK3, PIM1, CDC7L1, CDK7, CNK, PRL-3, STK2(NEK4), NKIAMRE, or HBO1 is achieved when the activity value relative tothe control is about 80%, preferably 50%, more preferably 25-0%.Activation of PKC-ζ, PLC-β1, FAK, FAK2, CK2, cMET, FEN1, REV1, APE1,CDK3, PIM1, CDC7L1, CDK7, CNK, PRL-3, STK2 (NEK4), NKIAMRE, or HBO1 isachieved when the activity value relative to the control (untreated withactivators) is 110%, more preferably 150%, more preferably 200-500%(i.e., two to five fold higher relative to the control), more preferably1000-3000% higher.

[0121] The term “test compound” or “drug candidate” or “modulator” orgrammatical equivalents as used herein describes any molecule, eithernaturally occurring or synthetic, e.g., protein, oligopeptide (e.g.,from about 5 to about 25 amino acids in length, preferably from about 10to 20 or 12 to 18 amino acids in length, preferably 12, 15, or 18 aminoacids in length), small organic molecule, polysaccharide, lipid, fattyacid, polynucleotide, oligonucleotide, etc., to be tested for thecapacity to directly or indirectly modulation tumor cell proliferation.The test compound can be in the form of a library of test compounds,such as a combinatorial or randomized library that provides a sufficientrange of diversity. Test compounds are optionally linked to a fusionpartner, e.g., targeting compounds, rescue compounds, dimerizationcompounds, stabilizing compounds, addressable compounds, and otherfunctional moieties. Conventionally, new chemical entities with usefulproperties are generated by identifying a test compound (called a “leadcompound”) with some desirable property or activity, e.g., inhibitingactivity, creating variants of the lead compound, and evaluating theproperty and activity of those variant compounds. Often, high throughputscreening (HTS) methods are employed for such an analysis.

[0122] A “small organic molecule” refers to an organic molecule, eithernaturally occurring or synthetic, that has a molecular weight of morethan about 50 daltons and less than about 2500 daltons, preferably lessthan about 2000 daltons, preferably between about 100 to about 1000daltons, more preferably between about 200 to about 500 daltons.

[0123] An “siRNA” refers to a nucleic acid that forms a double strandedRNA, which double stranded RNA has the ability to reduce or inhibitexpression of a gene or target gene when the siRNA expressed in the samecell as the gene or target gene. “siRNA” thus refers to the doublestranded RNA formed by the complementary strands. siRNA molecule andRNAi molecule are used interchangeably herein. The complementaryportions of the siRNA that hybridize to form the double strandedmolecule typically have substantial or complete identity. In oneembodiment, an siRNA refers to a nucleic acid that has substantial orcomplete identity to a target gene and forms a double stranded siRNA. Inanother embodiment, a “randomized siRNA” refers to a nucleic acid thatforms a double stranded siRNA, wherein the sequence of the siRNA israndomized. The sequence of the siRNA can correspond to the full lengthtarget gene, or a subsequence thereof. Typically, the siRNA is at leastabout 15-50 nucleotides in length (e.g., each complementary sequence ofthe double stranded siRNA is 15-50 nucleotides in length, and the doublestranded siRNA is about 15-50 base pairs in length, preferabley about15-30 nucleotides in length, preferably about 20-30 nucleotides inlength, preferably about 21-30 nucleotides in length, or about 20-25 orabout 24-29 nucleotides in length, e.g., 20, 21, 22, 23, 24, 25, 26, 27,28, 29, or 30 nucleotides in length.

[0124] “Biological sample” include sections of tissues such as biopsyand autopsy samples, and frozen sections taken for histologic purposes.Such samples include blood, sputum, tissue, cultured cells, e.g.,primary cultures, explants, and transformed cells, stool, urine, etc. Abiological sample is typically obtained from a eukaryotic organism, mostpreferably a mammal such as a primate e.g., chimpanzee or human; cow;dog; cat; a rodent, e.g., guinea pig, rat, mouse; rabbit; or a bird;reptile; or fish.

[0125] The terms “identical” or percent “identity,” in the context oftwo or more nucleic acids or polypeptide sequences, refer to two or moresequences or subsequences that are the same or have a specifiedpercentage of amino acid residues or nucleotides that are the same(i.e., about 60% identity, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over aspecified region (e.g., nucleotide sequence SEQ ID NO:1, 3, 5, 7, 9, 11,13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35 or amino acid sequenceSEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32,34, 36), when compared and aligned for maximum correspondence over acomparison window or designated region) as measured using a BLAST orBLAST 2.0 sequence comparison algorithms with default parametersdescribed below, or by manual aligmnent and visual inspection. Suchsequences are then said to be “substantially identical.” This definitionalso refers to, or may be applied to, the compliment of a test sequence.The definition also includes sequences that have deletions and/oradditions, as well as those that have substitutions. As described below,the preferred algorithms can account for gaps and the like. Preferably,identity exists over a region that is at least about 25 amino acids ornucleotides in length, or more preferably over a region that is 50-100amino acids or nucleotides in length.

[0126] For sequence comparison, typically one sequence acts as areference sequence, to which test sequences are compared. When using asequence comparison algorithm, test and reference sequences are enteredinto a computer, subsequence coordinates are designated, if necessary,and sequence algorithm program parameters are designated. Preferably,default program parameters can be used, or alternative parameters can bedesignated. The sequence comparison algorithm then calculates thepercent sequence identities for the test sequences relative to thereference sequence, based on the program parameters.

[0127] A “comparison window”, as used herein, includes reference to asegment of any one of the number of contiguous positions selected fromthe group consisting of from 20 to 600, usually about 50 to about 200,more usually about 100 to about 150 in which a sequence may be comparedto a reference sequence of the same number of contiguous positions afterthe two sequences are optimally aligned. Methods of alignment ofsequences for comparison are well-known in the art. Optimal alignment ofsequences for comparison can be conducted, e.g., by the local homologyalgorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by thehomology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443(1970), by the search for similarity method of Pearson & Lipman, Proc.Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized implementations ofthese algorithms (GAP, BESTFIT, FASTA, and TFASTA in the WisconsinGenetics Software Package, Genetics Computer Group, 575 Science Dr.,Madison, Wis.), or by manual alignment and visual inspection (see, e.g.,Current Protocols in Molecular Biology (Ausubel et al., eds. 1995supplement)).

[0128] A preferred example of algorithm that is suitable for determiningpercent sequence identity and sequence similarity are the BLAST andBLAST 2.0 algorithms, which are described in Altschul et al., Nuc. AcidsRes. 25:3389-3402 (1977) and Altschul et al., J. Mol. Biol. 215:403-410(1990), respectively. BLAST and BLAST 2.0 are used, with the parametersdescribed herein, to determine percent sequence identity for the nucleicacids and proteins of the invention. Software for performing BLASTanalyses is publicly available through the National Center forBiotechnology Information. This algorithm involves first identifyinghigh scoring sequence pairs (HSPs) by identifying short words of lengthW in the query sequence, which either match or satisfy somepositive-valued threshold score T when aligned with a word of the samelength in a database sequence. T is referred to as the neighborhood wordscore threshold (Altschul et al., supra). These initial neighborhoodword hits act as seeds for initiating searches to find longer HSPscontaining them. The word hits are extended in both directions alongeach sequence for as far as the cumulative alignment score can beincreased. Cumulative scores are calculated using, for nucleotidesequences, the parameters M (reward score for a pair of matchingresidues; always >0) and N (penalty score for mismatching residues;always <0). For amino acid sequences, a scoring matrix is used tocalculate the cumulative score. Extension of the word hits in eachdirection are halted when: the cumulative alignment score falls off bythe quantity X from its maximum achieved value; the cumulative scoregoes to zero or below, due to the accumulation of one or morenegative-scoring residue alignments; or the end of either sequence isreached. The BLAST algorithm parameters W, T, and X determine thesensitivity and speed of the alignment. The BLASTN program (fornucleotide sequences) uses as defaults a wordlength (W) of 11, anexpectation (E) of 10, M=5, N=−4 and a comparison of both strands. Foramino acid sequences, the BLASTP program uses as defaults a wordlengthof 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (seeHenikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989))alignments (B) of 50, expectation (E) of 10, M=5, N=−4, and a comparisonof both strands.

[0129] “Nucleic acid” refers to deoxyribonucleotides or ribonucleotidesand polymers thereof in either single- or double-stranded form. The termencompasses nucleic acids containing known nucleotide analogs ormodified backbone residues or linkages, which are synthetic, naturallyoccurring, and non-naturally occurring, which have similar bindingproperties as the reference nucleic acid, and which are metabolized in amanner similar to the reference nucleotides. Examples of such analogsinclude, without limitation, phosphorothioates, phosphoramidates, methylphosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides,peptide-nucleic acids (PNAs).

[0130] Unless otherwise indicated, a particular nucleic acid sequencealso implicitly encompasses conservatively modified variants thereof(e.g., degenerate codon substitutions) and complementary sequences, aswell as the sequence explicitly indicated. Specifically, degeneratecodon substitutions may be achieved by generating sequences in which thethird position of one or more selected (or all) codons is substitutedwith mixed-base and/or deoxyinosine residues (Batzer et al., NucleicAcid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608(1985); Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)). The termnucleic acid is used interchangeably with gene, cDNA, mRNA,oligonucleotide, and polynucleotide.

[0131] A particular nucleic acid sequence also implicitly encompasses“splice variants.” Similarly, a particular protein encoded by a nucleicacid implicitly encompasses any protein encoded by a splice variant ofthat nucleic acid. “Splice variants,” as the name suggests, are productsof alternative splicing of a gene. After transcription, an initialnucleic acid transcript may be spliced such that different (alternate)nucleic acid splice products encode different polypeptides. Mechanismsfor the production of splice variants vary, but include alternatesplicing of exons. Alternate polypeptides derived from the same nucleicacid by read-through transcription are also encompassed by thisdefinition. Any products of a splicing reaction, including recombinantforms of the splice products, are included in this definition. Anexample of potassium channel splice variants is discussed in Leicher, etal., J. Biol. Chem. 273(52):35095-35101 (1998).

[0132] The terms “polypeptide,” “eptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical mimetic of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers and non-naturally occurring amino acid polymer.

[0133] The term “amino acid” refers to naturally occurring and syntheticamino acids, as well as amino acid analogs and amino acid mimetics thatfunction in a manner similar to the naturally occurring amino acids.Naturally occurring amino acids are those encoded by the genetic code,as well as those amino acids that are later modified, e.g.,hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acidanalogs refers to compounds that have the same basic chemical structureas a naturally occurring amino acid, i.e., an α carbon that is bound toa hydrogen, a carboxyl group, an amino group, and an R group, e.g.,homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) ormodified peptide backbones, but retain the same basic chemical structureas a naturally occurring amino acid. Amino acid mimetics refers tochemical compounds that have a structure that is different from thegeneral chemical structure of an amino acid, but that functions in amanner similar to a naturally occurring amino acid.

[0134] Amino acids may be referred to herein by either their commonlyknown three letter symbols or by the one-letter symbols recommended bythe IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides,likewise, may be referred to by their commonly accepted single-lettercodes.

[0135] “Conservatively modified variants” applies to both amino acid andnucleic acid sequences. With respect to particular nucleic acidsequences, conservatively modified variants refers to those nucleicacids which encode identical or essentially identical amino acidsequences, or where the nucleic acid does not encode an amino acidsequence, to essentially identical sequences. Because of the degeneracyof the genetic code, a large number of functionally identical nucleicacids encode any given protein. For instance, the codons GCA, GCC, GCGand GCU all encode the amino acid alanine. Thus, at every position wherean alanine is specified by a codon, the codon can be altered to any ofthe corresponding codons described without altering the encodedpolypeptide. Such nucleic acid variations are “silent variations,” whichare one species of conservatively modified variations. Every nucleicacid sequence herein which encodes a polypeptide also describes everypossible silent variation of the nucleic acid. One of skill willrecognize that each codon in a nucleic acid (except AUG, which isordinarily the only codon for methionine, and TGG, which is ordinarilythe only codon for tryptophan) can be modified to yield a functionallyidentical molecule. Accordingly, each silent variation of a nucleic acidwhich encodes a polypeptide is implicit in each described sequence withrespect to the expression product, but not with respect to actual probesequences.

[0136] As to amino acid sequences, one of skill will recognize thatindividual substitutions, deletions or additions to a nucleic acid,peptide, polypeptide, or protein sequence which alters, adds or deletesa single amino acid or a small percentage of amino acids in the encodedsequence is a “conservatively modified variant” where the alterationresults in the substitution of an amino acid with a chemically similaramino acid. Conservative substitution tables providing funictionallysimilar amino acids are well known in the art. Such conservativelymodified variants are in addition to and do not exclude polymorphicvariants, interspecies homologs, and alleles of the invention.

[0137] The following eight groups each contain amino acids that areconservative substitutions for one another: 1) Alanine (A), Glycine (G);2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine(Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L),Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y),Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C),Methionine (M) (see, e.g., Creighton, Proteins (1984)).

[0138] Macromolecular structures such as polypeptide structures can bedescribed in terms of various levels of organization. For a generaldiscussion of this organization, see, e.g., Alberts et al., MolecularBiology of the Cell (3^(rd) ed., 1994) and Cantor and Schimmel,Biophysical Chemistry Part I: The Conformation of BiologicalMacromolecules (1980). “Primary structure” refers to the amino acidsequence of a particular peptide. “Secondary structure” refers tolocally ordered, three dimensional structures within a polypeptide.These structures are commonly known as domains, e.g., enzymatic domains,extracellular domains, transmembrane domains, pore domains, andcytoplasmic tail domains. Domains are portions of a polypeptide thatform a compact unit of the polypeptide and are typically 15 to 350 aminoacids long. Exemplary domains include domains with enzymatic activity,e.g., a kinase domain. Typical domains are made up of sections of lesserorganization such as stretches of β-sheet and α-helices. “Tertiarystructure” refers to the complete three dimensional structure of apolypeptide monomer. “Quaternary structure” refers to the threedimensional structure formed by the noncovalent association ofindependent tertiary units. Anisotropic terms are also known as energyterms.

[0139] A “label” or a “detectable moiety” is a composition detectable byspectroscopic, photochemical, biochemical, immunochemical, chemical, orother physical means. For example, useful labels include ³²P,fluorescent dyes, electron-dense reagents, enzymes (e.g., as commonlyused in an ELISA), biotin, digoxigenin, or haptens and proteins whichcan be made detectable, e.g., by incorporating a radiolabel into thepeptide or used to detect antibodies specifically reactive with thepeptide.

[0140] The term “recombinant” when used with reference, e.g., to a cell,or nucleic acid, protein, or vector, indicates that the cell, nucleicacid, protein or vector, has been modified by the introduction of aheterologous nucleic acid or protein or the alteration of a nativenucleic acid or protein, or that the cell is derived from a cell somodified. Thus, for example, recombinant cells express genes that arenot found within the native (non-recombinant) form of the cell orexpress native genes that are otherwise abnormally expressed, underexpressed or not expressed at all.

[0141] The term “heterologous” when used with reference to portions of anucleic acid indicates that the nucleic acid comprises two or moresubsequences that are not found in the same relationship to each otherin nature. For instance, the nucleic acid is typically recombinantlyproduced, having two or more sequences from unrelated genes arranged tomake a new functional nucleic acid, e.g., a promoter from one source anda coding region from another source. Similarly, a heterologous proteinindicates that the protein comprises two or more subsequences that arenot found in the same relationship to each other in nature (e.g., afusion protein).

[0142] The phrase “stringent hybridization conditions” refers toconditions under which a probe will hybridize to its target subsequence,typically in a complex mixture of nucleic acids, but to no othersequences. Stringent conditions are sequence-dependent and will bedifferent in different circumstances. Longer sequences hybridizespecifically at higher temperatures. An extensive guide to thehybridization of nucleic acids is found in Tijssen, Techniques inBiochemistry and Molecular Biology—Hybridization with Nucleic Probes,“Overview of principles of hybridization and the strategy of nucleicacid assays” (1993). Generally, stringent conditions are selected to beabout 5-10° C. lower than the thermal melting point (T_(m)) for thespecific sequence at a defined ionic strength pH. The T_(m) is thetemperature (under defined ionic strength, pH, and nucleicconcentration) at which 50% of the probes complementary to the targethybridize to the target sequence at equilibrium (as the target sequencesare present in excess, at T_(m), 50% of the probes are occupied atequilibrium). Stringent conditions may also be achieved with theaddition of destabilizing agents such as formamide. For selective orspecific hybridization, a positive signal is at least two timesbackground, preferably 10 times background hybridization. Exemplarystringent hybridization conditions can be as following: 50% formamide,5×SSC, and 1% SDS, incubating at 42° C., or, 5×SSC, 1% SDS, incubatingat 65° C., with wash in 0.2×SSC, and 0.1% SDS at 65° C.

[0143] Nucleic acids that do not hybridize to each other under stringentconditions are still substantially identical if the polypeptides whichthey encode are substantially identical. This occurs, for example, whena copy of a nucleic acid is created using the maximum codon degeneracypermitted by the genetic code. In such cases, the nucleic acidstypically hybridize under moderately stringent hybridization conditions.Exemplary “moderately stringent hybridization conditions” include ahybridization in a buffer of 40% formamide, 1 M NaCl, 1% SDS at 37° C.,and a wash in 1×SSC at 45° C. A positive hybridization is at least twicebackground. Those of ordinary skill will readily recognize thatalternative hybridization and wash conditions can be utilized to provideconditions of similar stringency. Additional guidelines for determininghybridization parameters are provided in numerous reference, e.g., andCurrent Protocols in Molecular Biology, ed. Ausubel, et al.

[0144] For PCR, a temperature of about 36° C. is typical for lowstringency amplification, although annealing temperatures may varybetween about 32° C. and 48° C. depending on primer length. For highstringency PCR amplification, a temperature of about 62° C. is typical,although high stringency annealing temperatures can range from about 50°C. to about 65° C., depending on the primer length and specificity.Typical cycle conditions for both high and low stringency amplificationsinclude a denaturation phase of 90° C.-95° C. for 30 sec-2 min., anannealing phase lasting 30 sec-2 min., and an extension phase of about72° C. for 1-2 min. Protocols and guidelines for low and high stringencyamplification reactions are provided, e.g., in Innis et al. (1990) PCRProtocols, A Guide to Methods and Applications, Academic Press, Inc.N.Y.).

[0145] “Antibody” refers to a polypeptide comprising a framework regionfrom an immunoglobulin gene or fragments thereof that specifically bindsand recognizes an antigen. The recognized immunoglobulin genes includethe kappa, lambda, alpha, gamma, delta, epsilon, and mu constant regiongenes, as well as the myriad immunoglobulin variable region genes. Lightchains are classified as either kappa or lambda. Heavy chains areclassified as gamma, mu, alpha, delta, or epsilon, which in turn definethe immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.Typically, the antigen-binding region of an antibody will be mostcritical in specificity and affinity of binding.

[0146] An exemplary immunoglobulin (antibody) structural unit comprisesa tetramer. Each tetramer is composed of two identical pairs ofpolypeptide chains, each pair having one “light” (about 25 kD) and one“heavy” chain (about 50-70 kD). The N-terminus of each chain defines avariable region of about 100 to 110 or more amino acids primarilyresponsible for antigen recognition. The terms variable light chain(V_(L)) and variable heavy chain (V_(H)) refer to these light and heavychains respectively.

[0147] Antibodies exist, e.g., as intact immunoglobulins or as a numberof well-characterized fragments produced by digestion with variouspeptidases. Thus, for example, pepsin digests an antibody below thedisulfide linkages in the hinge region to produce F(ab)′₂, a dimer ofFab which itself is a light chain joined to V_(H)-C_(H)1 by a disulfidebond. The F(ab)′₂ may be reduced under mild conditions to break thedisulfide linkage in the hinge region, thereby converting the F(ab)′₂dimer into an Fab′ monomer. The Fab′ monomer is essentially Fab withpart of the hinge region (see Fundamental Immunology (Paul ed., 3d ed.1993). While various antibody fragments are defined in terms of thedigestion of an intact antibody, one of skill will appreciate that suchfragments may be synthesized de novo either chemically or by usingrecombinant DNA methodology. Thus, the term antibody, as used herein,also includes antibody fragments either produced by the modification ofwhole antibodies, or those synthesized de novo using recombinant DNAmethodologies (e.g., single chain Fv) or those identified using phagedisplay libraries (see, e.g., McCafferty et al., Nature 348:552-554(1990))

[0148] For preparation of antibodies, e.g., recombinant, monoclonal, orpolyclonal antibodies, many technique known in the art can be used (see,e.g., Kohler & Milstein, Nature 256:495-497 (1975); Kozbor et al.,Immunology Today 4: 72 (1983); Cole et al., pp. 77-96 in MonoclonalAntibodies and Cancer Therapy, Alan R. Liss, Inc. (1985); Coligan,Current Protocols in Immunology (1991); Harlow & Lane, Antibodies, ALaboratory Manual (1988); and Goding, Monoclonal Antibodies: Principlesand Practice (2d ed. 1986)). The genes encoding the heavy and lightchains of an antibody of interest can be cloned from a cell, e.g., thegenes encoding a monoclonal antibody can be cloned from a hybridoma andused to produce a recombinant monoclonal antibody. Gene librariesencoding heavy and light chains of monoclonal antibodies can also bemade from hybridoma or plasma cells. Random combinations of the heavyand light chain gene products generate a large pool of antibodies withdifferent antigenic specificity (see, e.g., Kuby, Immunology (3^(rd) ed.1997)). Techniques for the production of single chain antibodies orrecombinant antibodies (U.S. Pat. No. 4,946,778, U.S. Pat. No.4,816,567) can be adapted to produce antibodies to polypeptides of thisinvention. Also, transgenic mice, or other organisms such as othermammals, may be used to express humanized or human antibodies (see,e.g., U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126;5,633,425; 5,661,016, Marks et al., Bio/Technology 10:779-783 (1992);Lonberg et al., Nature 368:856-859 (1994); Morrison, Nature 368:812-13(1994); Fishwild et al., Nature Biotechnology 14:845-51 (1996);Neuberger, Nature Biotechnology 14:826 (1996); and Lonberg & Huszar,Intern. Rev. Immunol. 13:65-93 (1995)). Alternatively, phage displaytechnology can be used to identify antibodies and heteromeric Fabfragments that specifically bind to selected antigens (see, e.g.,McCafferty et al., Nature 348:552-554 (1990); Marks et al.,Biotechnology 10:779-783 (1992)). Antibodies can also be madebispecific, i.e., able to recognize two different antigens (see, e.g.,WO 93/08829, Traunecker et al., EMBO J. 10:3655-3659 (1991); and Sureshet al., Methods in Enzymology 121:210 (1986)). Antibodies can also beheteroconjugates, e.g., two covalently joined antibodies, orimmunotoxins (see, e.g., U.S. Pat. No. 4,676,980, WO 91/00360; WO92/200373; and EP 03089).

[0149] Methods for humanizing or primatizing non-human antibodies arewell known in the art. Generally, a humanized antibody has one or moreamino acid residues introduced into it from a source which is non-human.These non-human amino acid residues are often referred to as importresidues, which are typically taken from an import variable domain.Humanization can be essentially performed following the method of Winterand co-workers (see, e.g., Jones et al., Nature 321:522-525 (1986);Riechmann et al., Nature 332:323-327 (1988); Verhoeyen et al., Science239:1534-1536 (1988) and Presta, Curr. Op. Struct. Biol. 2:593-596(1992)), by substituting rodent CDRs or CDR sequences for thecorresponding sequences of a human antibody. Accordingly, such humanizedantibodies are chimeric antibodies (U.S. Pat. No. 4,816,567), whereinsubstantially less than an intact human variable domain has beensubstituted by the corresponding sequence from a non-human species. Inpractice, humanized antibodies are typically human antibodies in whichsome CDR residues and possibly some FR residues are substituted byresidues from analogous sites in rodent antibodies.

[0150] A “chimeric antibody” is an antibody molecule in which (a) theconstant region, or a portion thereof, is altered, replaced or exchangedso that the antigen binding site (variable region) is linked to aconstant region of a different or altered class, effector functionand/or species, or an entirely different molecule which confers newproperties to the chimeric antibody, e.g., an enzyme, toxin, hormone,growth factor, drug, etc.; or (b) the variable region, or a portionthereof, is altered, replaced or exchanged with a variable region havinga different or altered antigen specificity.

[0151] In one embodiment, the antibody is conjugated to an “effector”moiety. The effector moiety can be any number of molecules, includinglabeling moieties such as radioactive labels or fluorescent labels, orcan be a therapeutic moiety. In one aspect the antibody modulates theactivity of the protein.

[0152] The phrase “specifically (or selectively) binds” to an antibodyor “specifically (or selectively) immunoreactive with,” when referringto a protein or peptide, refers to a binding reaction that isdeterminative of the presence of the protein, often in a heterogeneouspopulation of proteins and other biologics. Thus, under designatedimmunoassay conditions, the specified antibodies bind to a particularprotein at least two times the background and more typically more than10 to 100 times background. Specific binding to an antibody under suchconditions requires an antibody that is selected for its specificity fora particular protein. For example, polyclonal antibodies raised to aPKC-ζ, PLC-β1, FAK, FAK2, CK2, cMET, FEN1, REV1, APE1, CDK3, PIM1,CDC7L1, CDK7, CNK, PRL-3, STK2 (NEK4), NKIAMRE, or HBO1 protein,polymorphic variants, alleles, orthologs, and conservatively modifiedvariants, or splice variants, or portions thereof, can be selected toobtain only those polyclonal antibodies that are specificallyimmunoreactive with PKC-ζ, PLC-β1, FAK, FAK2, CK2, cMET, FEN1, REV1,APE1, CDK3, PIM1, CDC7L1, CDK7, CNK, PRL-3, STK2 (NEK4), NKIAMRE, orHBO1 proteins and not with other proteins. This selection may beachieved by subtracting out antibodies that cross-react with othermolecules. A variety of immunoassay formats may be used to selectantibodies specifically immunoreactive with a particular protein. Forexample, solid-phase ELISA immunoassays are routinely used to selectantibodies specifically immunoreactive with a protein (see, e.g., Harlow& Lane, Antibodies, A Laboratory Manual (1988) for a description ofimmunoassay formats and conditions that can be used to determinespecific immunoreactivity).

[0153] By “therapeutically effective dose” herein is meant a dose thatproduces effects for which it is administered. The exact dose willdepend on the purpose of the treatment, and will be ascertainable by oneskilled in the art using known techniques (see, e.g., Lieberman,Pharmaceutical Dosage Forms (vols. 1-3, 1992); Lloyd, The Art, Scienceand Technology of Pharmaceutical Compounding (1999); and Pickar, DosageCalculations (1999)).

[0154] Assays for Proteins that Modulate Cellular Proliferation

[0155] High throughput functional genomics assays can be used toidentify modulators of cellular proliferation. Such assays can monitorchanges in cell surface marker expression, proliferation anddifferentiation, and apoptosis, using either cell lines or primarycells. Typically, the cells are contacted with a cDNA or a randompeptide library (encoded by nucleic acids). In one embodiment, thepeptides are cyclic or circular. The cDNA library can comprise sense,antisense, full length, and truncated cDNAs. The peptide library isencoded by nucleic acids. The effect of the cDNA or peptide library onthe phenotype of cellular proliferation is then monitored, using anassay as described above. The effect of the cDNA or peptide can bevalidated and distinguished from somatic mutations, using, e.g.,regulatable expression of the nucleic acid such as expression from atetracycline promoter. cDNAs and nucleic acids encoding peptides can berescued using techniques known to those of skill in the art, e.g., usinga sequence tag.

[0156] Proteins interacting with the peptide or with the protein encodedby the cDNA (e.g., PKC-ζ, PLC-β1, FAK, FAK2, CK2, cMET, FEN1, REV1,APE1, CDK3, PIM1, CDC7L1, CDK7, CNK, PRL-3, STK2 (NEK4), NKIAMRE, orHBO1) can be isolated using a yeast two-hybrid system, mammalian twohybrid system, immunoprecipitation or affinity chromatography ofcomplexed proteins followed by mass spectrometry, or phage displayscreen, etc. Targets so identified can be further used as bait in theseassays to identify additional members of the cellular proliferationpathway, which members are also targets for drug development (see, e.g.,Fields et al., Nature 340:245 (1989); Vasavada et al., Proc. Nat 'lAcad. Sci. USA 88:10686 (1991); Fearon et al., Proc. Nat'l Acad. Sci.USA 89:7958 (1992); Dang et al., Mol. Cell. Biol. 11:954 (1991); Chienet al., Proc. Nat 'l Acad. Sci. USA 9578 (1991); and U.S. Pat. Nos.5,283,173, 5,667,973, 5,468,614, 5,525,490, and 5,637,463).

[0157] Suitable cell lines include A549, HeLa, Colo205, H1299, MCF7,MDA-MB-231, PC3, HMEC, PrEC. Cell surface markers can be assayed usingfluorescently labeled antibodies and FACS. Cell proliferation can bemeasured using ³H-thymidine incorporation, cell count by dye inclusion,MTT assay, BrdU incorporation, Cell Tracker assay. Apoptosis can bemeasured using dye inclusion, or by assaying for DNA laddering,increases in intracellular calcium, or caspase activation. Growth factorproduction can be measured using an immunoassay such as ELISA.

[0158] cDNA libraries are made from any suitable source. Librariesencoding random peptides are made according to techniques well known tothose of skill in the art (see, e.g., U.S. Pat. Nos. 6,153,380,6,114,111, and 6,180,343). Any suitable vector can be used for the cDNAand peptide libraries, including, e.g., retroviral vectors.

[0159] Isolation of Nucleic Acids Encoding PKC-ζ, PLC-β1, FAK, FAK2,CK2, cMET, FEN1, REV1, APE1, CDK3, PIM1, CDC7L1, CDK7, CNK, PRL-3, STK2(NEK4), NKIAMRE, or HBO1 Family Members

[0160] This invention relies on routine techniques in the field ofrecombinant genetics. Basic texts disclosing the general methods of usein this invention include Sambrook et al., Molecular Cloning, ALaboratory Manual (2nd ed. 1989); Kriegler, Gene Transfer andExpression: A Laboratory Manual (1990); and Current Protocols inMolecular Biology (Ausubel et al., eds., 1994)).

[0161] PKC-ζ, PLC-β1, FAK, FAK2, CK2, cMET, FEN1, REV1, APE1, CDK3,PIM1, CDC7L1, CDK7, CNK, PRL-3, STK2 (NEK4), NKIAMRE, or HBO1 nucleicacids, polymorphic variants, orthologs, and alleles that aresubstantially identical to an amino acid sequence encoded by SEQ IDNO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, or 36can be isolated using PKC-ζ, PLC-β1, FAK, FAK2, CK2, cMET, FEN1, REV1,APE1, CDK3, PIM1, CDC7L1, CDK7, CNK, PRL-3, STK2 (NEK4), NKIAMRE, orHBO1 nucleic acid probes and oligonucleotides under stringenthybridization conditions, by screening libraries. Alternatively,expression libraries can be used to clone PKC-ζ, PLC-β1, FAK, FAK2, CK2,cMET, FEN1, REV1, APE1, CDK3, PIM1, CDC7L1, CDK7, CNK, PRL-3, STK2(NEK4), NKIAMRE, or HBO1 protein, polymorphic variants, orthologs, andalleles by detecting expressed homologs immunologically with antisera orpurified antibodies made against human PKC-ζ, PLC-β1, FAK, FAK2, CK2,cMET, FEN1, REV1, APE1, CDK3, PIM1, CDC7L1, CDK7, CNK, PRL-3, STK2(NEK4), NKIAMRE, or HBO1 or portions thereof.

[0162] To make a cDNA library, one should choose a source that is richin PKC-ζ, PLC-β1, FAK, FAK2, CK2, cMET, FEN1, REV1, APE1, CDK3, PIM1,CDC7L1, CDK7, CNK, PRL-3, STK2 (NEK4), NKIAMRE, or HBO1 RNA. The mRNA isthen made into cDNA using reverse transcriptase, ligated into arecombinant vector, and transfected into a recombinant host forpropagation, screening and cloning. Methods for making and screeningcDNA libraries are well known (see, e.g., Gubler & Hoffman, Gene25:263-269 (1983); Sambrook et al., supra; Ausubel et al., supra).

[0163] For a genomic library, the DNA is extracted from the tissue andeither mechanically sheared or enzymatically digested to yield fragmentsof about 12-20 kb. The fragments are then separated by gradientcentrifugation from undesired sizes and are constructed in bacteriophagelambda vectors. These vectors and phage are packaged in vitro.Recombinant phage are analyzed by plaque hybridization as described inBenton & Davis, Science 196:180-182 (1977). Colony hybridization iscarried out as generally described in Grunstein et al., Proc. Natl.Acad. Sci. USA., 72:3961-3965 (1975).

[0164] An alternative method of isolating PKC-ζ, PLC-β1, FAK, FAK2, CK2,cMET, FEN1, REV1, APE1, CDK3, PIM1, CDC7L1, CDK7, CNK, PRL-3, STK2(NEK4), NKIAMRE, or HBO1 nucleic acid and its orthologs, alleles,mutants, polymorphic variants, and conservatively modified variantscombines the use of synthetic oligonucleotide primers and amplificationof an RNA or DNA template (see U.S. Pat. Nos. 4,683,195 and 4,683,202;PCR Protocols: A Guide to Methods and Applications (Innis et al., eds,1990)). Methods such as polymerase chain reaction (PCR) and ligase chainreaction (LCR) can be used to amplify nucleic acid sequences of humanPKC-ζ, PLC-β1, FAK, FAK2, CK2, cMET, FEN1, REV1, APE1, CDK3, PIM1,CDC7L1, CDK7, CNK, PRL-3, STK2 (NEK4), NKIAMRE, or HBO1 directly frommRNA, from cDNA, from genomic libraries or cDNA libraries. Degenerateoligonucleotides can be designed to amplify PKC-ζ, PLC-β1, FAK, FAK2,CK2, cMET, FEN1, REV1, APE1, CDK3, PIM1, CDC7L1, CDK7, CNK, PRL-3, STK2(NEK4), NKIAMRE, or HBO1 homologs using the sequences provided herein.Restriction endonuclease sites can be incorporated into the primers.Polymerase chain reaction or other in vitro amplification methods mayalso be useful, for example, to clone nucleic acid sequences that codefor proteins to be expressed, to make nucleic acids to use as probes fordetecting the presence of PKC-ζ, PLC-β1, FAK, FAK2, CK2, cMET, FEN1,REV1, APE1, CDK3, PIM1, CDC7L1, CDK7, CNK, PRL-3, STK2 (NEK4), NKIAMRE,or HBO1 encoding mRNA in physiological samples, for nucleic acidsequencing, or for other purposes. Genes amplified by the PCR reactioncan be purified from agarose gels and cloned into an appropriate vector.

[0165] Gene expression of PKC-ζ, PLC-β1, FAK, FAK2, CK2, cMET, FEN1,REV1, APE1, CDK3, PIM1, CDC7L1, CDK7, CNK, PRL-3, STK2 (NEK4), NKIAMRE,or HBO1 can also be analyzed by techniques known in the art, e.g.,reverse transcription and amplification of mRNA, isolation of total RNAor poly A⁺ RNA, northern blotting, dot blotting, in situ hybridization,RNase protection, high density polynucleotide array technology, e.g.,and the like.

[0166] Nucleic acids encoding PKC-ζ, PLC-β1, FAK, FAK2, CK2, cMET, FEN1,REV1, APE1, CDK3, PIM1, CDC7L1, CDK7, CNK, PRL-3, STK2 (NEK4), NKIAMRE,or HBO1 protein can be used with high density oligonucleotide arraytechnology (e.g., GeneChip™) to identify PKC-ζ, PLC-β1, FAK, FAK2, CK2,cMET, FEN1, REV1, APE1, CDK3, PIM1, CDC7L1, CDK7, CNK, PRL-3, STK2(NEK4), NKIAMRE, or HBO1 protein, orthologs, alleles, conservativelymodified variants, and polymorphic variants in this invention. In thecase where the homologs being identified are linked to modulation ofcellular proliferation, they can be used with GeneChip™ as a diagnostictool in detecting the disease in a biological sample, see, e.g.,Gunthand et al., AIDS Res. Hum. Retroviruses 14: 869-876 (1998); Kozalet al., Nat. Med. 2:753-759 (1996); Matson et al., Anal. Biochem.224:110-106 (1995); Lockhart et al., Nat. Biotechnol. 14:1675-1680(1996); Gingeras et al., Genome Res. 8:435-448 (1998); Hacia et al.,Nucleic Acids Res. 26:3865-3866 (1998).

[0167] The gene for PKC-ζ, PLC-β1, FAK, FAK2, CK2, cMET, FEN1, REV1,APE1, CDK3, PIM1, CDC7L1, CDK7, CNK, PRL-3, STK2 (NEK4), NKIAMRE, orHBO1 is typically cloned into intermediate vectors before transformationinto prokaryotic or eukaryotic cells for replication and/or expression.These intermediate vectors are typically prokaryote vectors, e.g.,plasmids, or shuttle vectors.

[0168] Expression in Prokaryotes and Eukaryotes

[0169] To obtain high level expression of a cloned gene, such as thosecDNAs encoding PKC-ζ, PLC-β1, FAK, FAK2, CK2, cMET, FEN1, REV1, APE1,CDK3, PIM1, CDC7L1, CDK7, CNK, PRL-3, STK2 (NEK4), NKIAMRE, or HBO1, onetypically subclones PKC-ζ, PLC-β1, FAK, FAK2, CK2, cMET, FEN1, REV1,APE1, CDK3, PIM1, CDC7L1, CDK7, CNK, PRL-3, STK2 (NEK4), NKIAMRE, orHBO1 into an expression vector that contains a strong promoter to directtranscription, a transcription/translation terminator, and if for anucleic acid encoding a protein, a ribosome binding site fortranslational initiation. Suitable bacterial promoters are well known inthe art and described, e.g., in Sambrook et al., and Ausubel et al,supra. Bacterial expression systems for expressing the PKC-ζ, PLC-β1,FAK, FAK2, CK2, cMET, FEN1, REV1, APE1, CDK3, PIM1, CDC7L1, CDK7, CNK,PRL-3, STK2 (NEK4), NKIAMRE, or HBO1 protein are available in, e.g., E.coli, Bacillus sp., and Salmonella (Palva et al., Gene 22:229-235(1983); Mosbach et al., Nature 302:543-545 (1983). Kits for suchexpression systems are commercially available. Eukaryotic expressionsystems for mammalian cells, yeast, and insect cells are well known inthe art and are also commercially available. In one preferredembodiment, retroviral expression systems are used in the presentinvention.

[0170] Selection of the promoter used to direct expression of aheterologous nucleic acid depends on the particular application. Thepromoter is preferably positioned about the same distance from theheterologous transcription start site as it is from the transcriptionstart site in its natural setting. As is known in the art, however, somevariation in this distance can be accommodated without loss of promoterfunction.

[0171] In addition to the promoter, the expression vector typicallycontains a transcription unit or expression cassette that contains allthe additional elements required for the expression of the PKC-ζ,PLC-β1, FAK, FAK2, CK2, cMET, FEN1, REV1, APE1, CDK3, PIM1, CDC7L1,CDK7, CNK, PRL-3, STK2 (NEK4), NKIAMRE, or HBO1 encoding nucleic acid inhost cells. A typical expression cassette thus contains a promoteroperably linked to the nucleic acid sequence encoding PKC-ζ, PLC-β1,FAK, FAK2, CK2, cMET, FEN1, REV1, APE1, CDK3, PIM1, CDC7L1, CDK7, CNK,PRL-3, STK2 (NEK4), NKIAMRE, or HBO1 and signals required for efficientpolyadenylation of the transcript, ribosome binding sites, andtranslation termination. Additional elements of the cassette may includeenhancers and, if genomic DNA is used as the structural gene, intronswith functional splice donor and acceptor sites.

[0172] In addition to a promoter sequence, the expression cassetteshould also contain a transcription termination region downstream of thestructural gene to provide for efficient termination. The terminationregion may be obtained from the same gene as the promoter sequence ormay be obtained from different genes.

[0173] The particular expression vector used to transport the geneticinformation into the cell is not particularly critical. Any of theconventional vectors used for expression in eukaryotic or prokaryoticcells may be used. Standard bacterial expression vectors includeplasmids such as pBR322 based plasmids, pSKF, pET23D, and fusionexpression systems such as MBP, GST, and LacZ. Epitope tags can also beadded to recombinant proteins to provide convenient methods ofisolation, e.g., c-myc. Sequence tags may be included in an expressioncassette for nucleic acid rescue. Markers such as fluorescent proteins,green or red fluorescent protein, β-gal, CAT, and the like can beincluded in the vectors as markers for vector transduction.

[0174] Expression vectors containing regulatory elements from eukaryoticviruses are typically used in eukaryotic expression vectors, e.g., SV40vectors, papilloma virus vectors, retroviral vectors, and vectorsderived from Epstein-Barr virus. Other exemplary eukaryotic vectorsinclude pMSG, pAV009/A⁺, pMTO10/A⁺, pMAMneo-5, baculovirus pDSVE, andany other vector allowing expression of proteins under the direction ofthe CMV promoter, SV40 early promoter, SV40 later promoter,metallothionein promoter, murine mammary tumor virus promoter, Roussarcoma virus promoter, polyhedrin promoter, or other promoters showneffective for expression in eukaryotic cells.

[0175] Expression of proteins from eukaryotic vectors can be also beregulated using inducible promoters. With inducible promoters,expression levels are tied to the concentration of inducing agents, suchas tetracycline or ecdysone, by the incorporation of response elementsfor these agents into the promoter. Generally, high level expression isobtained from inducible promoters only in the presence of the inducingagent; basal expression levels are minimal.

[0176] In one embodiment, the vectors of the invention have aregulatable promoter, e.g., tet-regulated systems and the RU-486 system(see, e.g., Gossen & Bujard, Proc. Nat'l Acad. Sci. USA 89:5547 (1992);Oligino et al., Gene Ther. 5:491-496 (1998); Wang et al., Gene Ther.4:432-441 (1997); Neering et al., Blood 88:1147-1155 (1996); and Rendahlet al., Nat. Biotechnol. 16:757-761 (1998)). These impart small moleculecontrol on the expression of the candidate target nucleic acids. Thisbeneficial feature can be used to determine that a desired phenotype iscaused by a transfected cDNA rather than a somatic mutation.

[0177] Some expression systems have markers that provide geneamplification such as thymidine kinase and dihydrofolate reductase.Alternatively, high yield expression systems not involving geneamplification are also suitable, such as using a baculovirus vector ininsect cells, with a PKC-ζ, PLC-β1, FAK, FAK2, CK2, cMET, FEN1, REV1,APE1, CDK3, PIM1, CDC7L1, CDK7, CNK, PRL-3, STK2 (NEK4), NKIAMRE, orHBO1 encoding sequence under the direction of the polyhedrin promoter orother strong baculovirus promoters.

[0178] The elements that are typically included in expression vectorsalso include a replicon that functions in E. coli , a gene encodingantibiotic resistance to permit selection of bacteria that harborrecombinant plasmids, and unique restriction sites in nonessentialregions of the plasmid to allow insertion of eukaryotic sequences. Theparticular antibiotic resistance gene chosen is not critical, any of themany resistance genes known in the art are suitable. The prokaryoticsequences are preferably chosen such that they do not interfere with thereplication of the DNA in eukaryotic cells, if necessary.

[0179] Standard transfection methods are used to produce bacterial,mammalian, yeast or insect cell lines that express large quantities ofPKC-ζ, PLC-β1, FAK, FAK2, CK2, cMET, FEN1, REV1, APE1, CDK3, PIM1,CDC7L1, CDK7, CNK, PRL-3, STK2 (NEK4), NKIAMRE, or HBO1 protein, whichare then purified using standard techniques (see, e.g., Colley et al.,J. Biol. Chem. 264:17619-17622 (1989); Guide to Protein Purification, inMethods in Enzymology, vol. 182 (Deutscher, ed., 1990)). Transformationof eukaryotic and prokaryotic cells are performed according to standardtechniques (see, e.g. Morrison, J. Bact. 132:349-351 (1977);Clark-Curtiss & Curtiss, Methods in Enzymology 101:347-362 (Wu et al.,eds, 1983).

[0180] Any of the well-known procedures for introducing foreignnucleotide sequences into host cells may be used. These include the useof calcium phosphate transfection, polybrene, protoplast fusion,electroporation, biolistics, liposomes, microinjection, plasma vectors,viral vectors and any of the other well known methods for introducingcloned genomic DNA, cDNA, synthetic DNA or other foreign geneticmaterial into a host cell (see, e.g., Sambrook et al., supra). It isonly necessary that the particular genetic engineering procedure used becapable of successfully introducing at least one gene into the host cellcapable of expressing PKC-ζ, PLC-β1, FAK, FAK2, CK2, cMET, FEN1, REV1,APE1, CDK3, PIM1, CDC7L1, CDK7, CNK, PRL-3, STK2 (NEK4), NKIAMRE, orHBO1.

[0181] After the expression vector is introduced into the cells, thetransfected cells are cultured under conditions favoring expression ofPKC-ζ, PLC-β1, FAK, FAK2, CK2, cMET, FEN1, REV1, APE1, CDK3, PIM1,CDC7L1, CDK7, CNK, PRL-3, STK2 (NEK4), NKIAMRE, or HBO1, which isrecovered from the culture using standard techniques identified below.

[0182] Purification of PKC-ζ, PLC-β1, FAK, FAK2, CK2, cMET, FEN1, REV1,APE1, CDK3, PIM1, CDC7L1, CDK7, CNK, PRL-3, STK2 (NEK4), NKIAMRE, orHBO1 Polypeptides

[0183] Either naturally occurring or recombinant PKC-ζ, PLC-β1, FAK,FAK2, CK2, cMET, FEN1, REV1, APE1, CDK3, PIM1, CDC7L1, CDK7, CNK, PRL-3,STK2 (NEK4), NKIAMRE, or HBO1 can be purified for use in functionalassays. Naturally occurring PKC-ζ, PLC-β1, FAK, FAK2, CK2, cMET, FEN1,REV1, APE1, CDK3, PIM1, CDC7L1, CDK7, CNK, PRL-3, STK2 (NEK4), NKIAMRE,or HBO1 can be purified, e.g., from human tissue. Recombinant PKC-ζ,PLC-β1, FAK, FAK2, CK2, cMET, FEN1, REV1, APE1, CDK3, PIM1, CDC7L1,CDK7, CNK, PRL-3, STK2 (NEK4), NKIAMRE, or HBO1 can be purified from anysuitable expression system.

[0184] The PKC-ζ, PLC-β1, FAK, FAK2, CK2, cMET, FEN1, REV1, APE1, CDK3,PIM1, CDC7L1, CDK7, CNK, PRL-3, STK2 (NEK4), NKIAMRE, or HBO1 proteinmay be purified to substantial purity by standard techniques, includingselective precipitation with such substances as ammonium sulfate; columnchromatography, immunopurification methods, and others (see, e.g.,Scopes, Protein Purification: Principles and Practice (1982); U.S. Pat.No. 4,673,641; Ausubel et al., supra; and Sambrook et al.; supra).

[0185] A number of procedures can be employed when recombinant PKC-ζ,PLC-β1, FAK, FAK2, CK2, cMET, FEN1, REV1, APE1, CDK3, PIM1, CDC7L1,CDK7, CNK, PRL-3, STK2 (NEK4), NKIAMRE, or HBO1 protein is beingpurified. For example, proteins having established molecular adhesionproperties can be reversible fused to the PKC-ζ, PLC-β1, FAK, FAK2, CK2,cMET, FEN1, REV1, APE1, CDK3, PIM1, CDC7L1, CDK7, CNK, PRL-3, STK2(NEK4), NKIAMRE, or HBO1 protein. With the appropriate ligand orsubstrate, e.g., antiphospho S/T antibodies or anti-PKC-ζ, PLC-β1, FAK,FAK2, CK2, cMET, FEN1, REV1, APE1, CDK3, PIM1, CDC7L1, CDK7, CNK, PRL-3,STK2 (NEK4), NKIAMRE, or HBO1 antibodies, PKC-ζ, PLC-β1, FAK, FAK2, CK2,cMET, FEN1, REV1, APE1, CDK3, PIM1, CDC7L1, CDK7, CNK, PRL-3, STK2(NEK4), NKIAMRE, or HBO1 protein can be selectively adsorbed to apurification column and then freed from the column in a relatively pureform. The fused protein is then removed by enzymatic activity. Finally,PKC-ζ, PLC-β1, FAK, FAK2, CK2, cMET, FEN1, REV1, APE1, CDK3, PIM1,CDC7L1, CDK7, CNK, PRL-3, STK2 (NEK4), NKIAMRE, or HBO1 protein could bepurified using immunoaffinity columns. Recombinant PKC-ζ, PLC-β1, FAK,FAK2, CK2, cMET, FEN1, REV1, APE1, CDK3, PIM1, CDC7L1, CDK7, CNK, PRL-3,STK2 (NEK4), NKIAMRE, or HBO1 protein can be purified from any suitablesource, include yeast, insect, bacterial, and mammalian cells.

[0186] A. Purification of PKC-ζ, PLC-β1, FAK, FAK2, CK2, cMET, FEN1,REV1, APE1, CDK3, PIM1, CDC7L1, CDK7, CNK, PRL-3, STK2 (NEK4), NKIAMRE,or HBO1 from Recombinant Bacteria

[0187] Recombinant proteins are expressed by transformed bacteria inlarge amounts, typically after promoter induction; but expression can beconstitutive. Promoter induction with IPTG is one example of aninducible promoter system. Bacteria are grown according to standardprocedures in the art. Fresh or frozen bacteria cells are used forisolation of protein.

[0188] Proteins expressed in bacteria may form insoluble aggregates(“inclusion bodies”). Several protocols are suitable for purification ofPKC-ζ, PLC-β1, FAK, FAK2, CK2, cMET, FEN1, REV1, APE1, CDK3, PIM1,CDC7L1, CDK7, CNK, PRL-3, STK2 (NEK4), NKIAMRE, or HBO1 proteininclusion bodies. For example, purification of inclusion bodiestypically involves the extraction, separation and/or purification ofinclusion bodies by disruption of bacterial cells, e.g., by incubationin a buffer of 50 mM TRIS/HCL pH 7.5, 50 mM NaCl, 5 mM MgCl₂, 1 mM DTT,0.1 mM ATP, and 1 mM PMSF. The cell suspension can be lysed using 2-3passages through a French Press, homogenized using a Polytron (BrinkmanInstruments) or sonicated on ice. Alternate methods of lysing bacteriaare apparent to those of skill in the art (see, e.g., Sambrook et al.,supra; Ausubel et al., supra).

[0189] If necessary, the inclusion bodies are solubilized, and the lysedcell suspension is typically centrifuged to remove unwanted insolublematter. Proteins that formed the inclusion bodies may be renatured bydilution or dialysis with a compatible buffer. Suitable solventsinclude, but are not limited to urea (from about 4 M to about 8 M),formamide (at least about 80%, volume/volume basis), and guanidinehydrochloride (from about 4 M to about 8 M). Some solvents which arecapable of solubilizing aggregate-forming proteins, for example SDS(sodium dodecyl sulfate), 70% formic acid, are inappropriate for use inthis procedure due to the possibility of irreversible denaturation ofthe proteins, accompanied by a lack of immunogenicity and/or activity.Although guanidine hydrochloride and similar agents are denaturants,this denaturation is not irreversible and renaturation may occur uponremoval (by dialysis, for example) or dilution of the denaturant,allowing re-formation of immunologically and/or biologically activeprotein. Other suitable buffers are known to those skilled in the art.Human PKC-ζ, PLC-β1, FAK, FAK2, CK2, cMET, FEN1, REV1, APE1, CDK3, PIM1,CDC7L1, CDK7, CNK, PRL-3, STK2 (NEK4), NKIAMRE, or HBO1 proteins areseparated from other bacterial proteins by standard separationtechniques, e.g., with Ni—NTA agarose resin.

[0190] Alternatively, it is possible to purify PKC-ζ, PLC-β1, FAK, FAK2,CK2, cMET, FEN1, REV1, APE1, CDK3, PIM1, CDC7L1, CDK7, CNK, PRL-3, STK2(NEK4), NKIAMRE, or HBO1 protein from bacteria periplasm. After lysis ofthe bacteria, when the PKC-ζ, PLC-β1, FAK, FAK2, CK2, cMET, FEN1, REV1,APE1, CDK3, PIM1, CDC7L1, CDK7, CNK, PRL-3, STK2 (NEK4), NKIAMRE, orHBO1 protein exported into the periplasm of the bacteria, theperiplasmic fraction of the bacteria can be isolated by cold osmoticshock in addition to other methods known to skill in the art. To isolaterecombinant proteins from the periplasm, the bacterial cells arecentrifuged to form a pellet. The pellet is resuspended in a buffercontaining 20% sucrose. To lyse the cells, the bacteria are centrifugedand the pellet is resuspended in ice-cold 5 mM MgSO₄ and kept in an icebath for approximately 10 minutes. The cell suspension is centrifugedand the supernatant decanted and saved. The recombinant proteins presentin the supernatant can be separated from the host proteins by standardseparation techniques well known to those of skill in the art.

[0191] B. Standard Protein Separation Techniques for Purifying PKC-ζ,PLC-β1, FAK, FAK2, CK2, cMET, FEN1, REV1, APE1, CDK3, PIM1, CDC7L1,CDK7, CNK, PRL-3, STK2 (NEK4), NKIAMRE, or HBO1 proteins

[0192] Solubility Fractionation

[0193] Often as an initial step, particularly if the protein mixture iscomplex, an initial salt fractionation can separate many of the unwantedhost cell proteins (or proteins derived from the cell culture media)from the recombinant protein of interest. The preferred salt is ammoniumsulfate. Ammonium sulfate precipitates proteins by effectively reducingthe amount of water in the protein mixture. Proteins then precipitate onthe basis of their solubility. The more hydrophobic a protein is, themore likely it is to precipitate at lower ammonium sulfateconcentrations. A typical protocol includes adding saturated ammoniumsulfate to a protein solution so that the resultant ammonium sulfateconcentration is between 20-30%. This concentration will precipitate themost hydrophobic of proteins. The precipitate is then discarded (unlessthe protein of interest is hydrophobic) and ammonium sulfate is added tothe supernatant to a concentration known to precipitate the protein ofinterest. The precipitate is then solubilized in buffer and the excesssalt removed if necessary, either through dialysis or diafiltration.Other methods that rely on solubility of proteins, such as cold ethanolprecipitation, are well known to those of skill in the art and can beused to fractionate complex protein mixtures.

[0194] Size Differential Filtration

[0195] The molecular weight of the PKC-ζ, PLC-β1, FAK, FAK2, CK2, cMET,FEN1, REV1, APE1, CDK3, PIM1, CDC7L1, CDK7, CNK, PRL-3, STK2 (NEK4),NKIAMRE, or HBO1 proteins can be used to isolate it from proteins ofgreater and lesser size using ultrafiltration through membranes ofdifferent pore size (for example, Amicon or Millipore membranes). As afirst step, the protein mixture is ultrafiltered through a membrane witha pore size that has a lower molecular weight cut-off than the molecularweight of the protein of interest. The retentate of the ultrafiltrationis then ultrafiltered against a membrane with a molecular cut offgreater than the molecular weight of the protein of interest. Therecombinant protein will pass through the membrane into the filtrate.The filtrate can then be chromatographed as described below.

[0196] Column Chromatography

[0197] The PKC-ζ, PLC-β1, FAK, FAK2, CK2, cMET, FEN1, REV1, APE1, CDK3,PIM1, CDC7L1, CDK7, CNK, PRL-3, STK2 (NEK4), NKIAMRE, or HBO1 proteinscan also be separated from other proteins on the basis of its size, netsurface charge, hydrophobicity, and affinity for ligands. In addition,antibodies raised against proteins can be conjugated to column matricesand the proteins immunopurified. All of these methods are well known inthe art. It will be apparent to one of skill that chromatographictechniques can be performed at any scale and using equipment from manydifferent manufacturers (e.g., Pharmacia Biotech).

[0198] Assays for Modulators of PKC-ζ, PLC-β1, FAK, FAK2, CK2, cMET,FEN1, REV1, APE1, CDK3, PIM1, CDC7L1, CDK7, CNK, PRL-3, STK2 (NEK4),NKIAMRE, or HBO1 Protein

[0199] A. Assays

[0200] Modulation of a PKC-ζ, PLC-β1, FAK, FAK2, CK2, cMET, FEN1, REV1,APE1, CDK3, PIM1, CDC7L1, CDK7, CNK, PRL-3, STK2 (NEK4), NKIAMRE, orHBO1 protein, and corresponding modulation of cellular, e.g., tumorcell, proliferation, can be assessed using a variety of in vitro and invivo assays, including cell-based models. Such assays can be used totest for inhibitors and activators of PKC-ζ, PLC-β1, FAK, FAK2, CK2,cMET, FEN1, REV1, APE1, CDK3, PIM1, CDC7L1, CDK7, CNK, PRL-3, STK2(NEK4), NKIAMRE, or HBO1 protein, and, consequently, inhibitors andactivators of cellular proliferation, including modulators ofchemotherapeutic sensitivity and toxicity. Such modulators of PKC-ζ,PLC-β1, FAK, FAK2, CK2, cMET, FEN1, REV1, APE1, CDK3, PIM1, CDC7L1,CDK7, CNK, PRL-3, STK2 (NEK4), NKIAMRE, or HBO1 protein are useful fortreating disorders related to pathological cell proliferation, e.g.,cancer. Modulators of PKC-ζ, PLC-β1, FAK, FAK2, CK2, cMET, FEN1, REV1,APE1, CDK3, PIM1, CDC7L1, CDK7, CNK, PRL-3, STK2 (NEK4), NKIAMRE, orHBO1 protein are tested using either recombinant or naturally occurringPKC-ζ, PLC-β1, FAK, FAK2, CK2, cMET, FEN1, REV1, APE1, CDK3, PIM1,CDC7L1, CDK7, CNK, PRL-3, STK2 (NEK4), NKIAMRE, or HBO1, preferablyhuman PKC-ζ, PLC-β1, FAK, FAK2, CK2, cMET, FEN1, REV1, APE1, CDK3, PIM1,CDC7L1, CDK7, CNK, PRL-3, STK2 (NEK4), NKIAMRE, or HBO1.

[0201] Preferably, the PKC-ζ, PLC-β1, FAK, FAK2, CK2, cMET, FEN1, REV1,APE1, CDK3, PIM1, CDC7L1, CDK7, CNK, PRL-3, STK2 (NEK4), NKIAMRE, orHBO1 protein will have the sequence as encoded by SEQ ID NO:2, 4, 6, 8,10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36 or aconservatively modified variant thereof. Alternatively, the PKC-ζ,PLC-β1, FAK, FAK2, CK2, cMET, FEN1, REV1, APE1, CDK3, PIM1, CDC7L1,CDK7, CNK, PRL-3, STK2 (NEK4), NKIAMRE, or HBO1 protein of the assaywill be derived from a eukaryote and include an amino acid subsequencehaving substantial amino acid sequence identity to SEQ ID NO:2, 4, 6, 8,10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36. Generally, theamino acid sequence identity will be at least 60%, preferably at least65%, 70%, 75%, 80%, 85%, or 90%, most preferably at least 95%.

[0202] Measurement of cellular proliferation modulation with PKC-ζ,PLC-β1, FAK, FAK2, CK2, cMET, FEN1, REV1, APE1, CDK3, PIM1, CDC7L1,CDK7, CNK, PRL-3, STK2 (NEK4), NKIAMRE, or HBO1 protein or a cellexpressing PKC-ζ, PLC-β1, FAK, FAK2, CK2, cMET, FEN1, REV1, APE1, CDK3,PIM1, CDC7L1, CDK7, CNK, PRL-3, STK2 (NEK4), NKIAMRE, or HBO1 protein,either recombinant or naturally occurring, can be performed using avariety of assays, in vitro, in vivo, and ex vivo, as described herein.A suitable physical, chemical or phenotypic change that affectsactivity, e.g., enzymatic activity such as kinase activity, cellproliferation, or ligand binding can be used to assess the influence ofa test compound on the polypeptide of this invention. When thefunctional effects are determined using intact cells or animals, one canalso measure a variety of effects, such as, ligand binding, kinaseactivity, transcriptional changes to both known and uncharacterizedgenetic markers (e.g., northern blots), changes in cell metabolism,changes related to cellular proliferation, cell surface markerexpression, DNA synthesis, marker and dye dilution assays (e.g., GFP andcell tracker assays), contact inhibition, tumor growth in nude mice,etc.

[0203] In Vitro Assays

[0204] Assays to identify compounds with PKC-ζ, PLC-β1, FAK, FAK2, CK2,cMET, FEN1, REV1, APE1, CDK3, PIM1, CDC7L1, CDK7, CNK, PRL-3, STK2(NEK4), NKIAMRE, or HBO1 modulating activity can be performed in vitro.Such assays can use full length PKC-ζ, PLC-β1, FAK, FAK2, CK2, cMET,FEN1, REV1, APE1, CDK3, PIM1, CDC7L1, CDK7, CNK, PRL-3, STK2 (NEK4),NKIAMRE, or HBO1 protein or a variant thereof (see, e.g., SEQ ID NO:2,4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36), or amutant thereof, or a fragment of a PKC-ζ, PLC-β1, FAK, FAK2, CK2, cMET,FEN1, REV1, APE1, CDK3, PIM1, CDC7L1, CDK7, CNK, PRL-3, STK2 (NEK4),NKIAMRE, or HBO1 protein, such as a kinase domain. Purified recombinantor naturally occurring PKC-ζ, PLC-β1, FAK, FAK2, CK2, cMET, FEN1, REV1,APE1, CDK3, PIM1, CDC7L1, CDK7, CNK, PRL-3, STK2 (NEK4), NKIAMRE, orHBO1 protein can be used in the in vitro methods of the invention. Inaddition to purified PKC-ζ, PLC-β1, FAK, FAK2, CK2, cMET, FEN1, REV1,APE1, CDK3, PIM1, CDC7L1, CDK7, CNK, PRL-3, STK2 (NEK4), NKIAMRE, orHBO1 protein, the recombinant or naturally occurring PKC-ζ, PLC-β1, FAK,FAK2, CK2, cMET, FEN1, REV1, APE1, CDK3, PIM1, CDC7L1, CDK7, CNK, PRL-3,STK2 (NEK4), NKIAMRE, or HBO1 protein can be part of a cellular lysateor a cell membrane. As described below, the binding assay can be eithersolid state or soluble. Preferably, the protein or membrane is bound toa solid support, either covalently or non-covalently. Often, the invitro assays of the invention are substrate or ligand binding oraffinity assays, either non-competitive or competitive. Other in vitroassays include measuring changes in spectroscopic (e.g., fluorescence,absorbance, refractive index), hydrodynamic (e.g., shape),chromatographic, or solubility properties for the protein. Other invitro assays include enzymatic activity assays, such as phosphorylationor autophosphorylation assays.

[0205] In one embodiment, a high throughput binding assay is performedin which the PKC-ζ, PLC-β1, FAK, FAK2, CK2, cMET, FEN1, REV1, APE1,CDK3, PIM1, CDC7L1, CDK7, CNK, PRL-3, STK2 (NEK4), NKIAMRE, or HBO1protein or a fragment thereof is contacted with a potential modulatorand incubated for a suitable amount of time. In one embodiment, thepotential modulator is bound to a solid support, and the PKC-ζ, PLC-β1,FAK, FAK2, CK2, cMET, FEN1, REV1, APE1, CDK3, PIM1, CDC7L1, CDK7, CNK,PRL-3, STK2 (NEK4), NKIAMRE, or HBO1 protein is added. In anotherembodiment, the PKC-ζ, PLC-β1, FAK, FAK2, CK2, cMET, FEN1, REV1, APE1,CDK3, PIM1, CDC7L1, CDK7, CNK, PRL-3, STK2 (NEK4), NKIAMRE, or HBO1protein is bound to a solid support. A wide variety of modulators can beused, as described below, including small organic molecules, peptides,antibodies, and PKC-ζ, PLC-β1, FAK, FAK2, CK2, cMET, FEN1, REV1, APE1,CDK3, PIM1, CDC7L1, CDK7, CNK, PRL-3, STK2 (NEK4), NKIAMRE, or HBO1ligand analogs. A wide variety of assays can be used to identify PKC-ζ,PLC-β1, FAK, FAK2, CK2, cMET, FEN1, REV1, APE1, CDK3, PIM1, CDC7L1,CDK7, CNK, PRL-3, STK2 (NEK4), NKIAMRE, or HBO1-modulator binding,including labeled protein-protein binding assays, electrophoreticmobility shifts, immunoassays, enzymatic assays such as kinase assays,and the like. In some cases, the binding of the candidate modulator isdetermined through the use of competitive binding assays, whereinterference with binding of a known ligand or substrate is measured inthe presence of a potential modulator. Either the modulator or the knownligand or substrate is bound first, and then the competitor is added.After the PKC-ζ, PLC-β1, FAK, FAK2, CK2, cMET, FEN1, REV1, APE1, CDK3,PIM1, CDC7L1, CDK7, CNK, PRL-3, STK2 (NEK4), NKIAMRE, or HBO1 protein iswashed, interference with binding, either of the potential modulator orof the known ligand or substrate, is determined. Often, either thepotential modulator or the known ligand or substrate is labeled.

[0206] Cell-Based In Vivo Assays

[0207] In another embodiment, PKC-ζ, PLC-β1, FAK, FAK2, CK2, cMET, FEN1,REV1, APE1, CDK3, PIM1, CDC7L1, CDK7, CNK, PRL-3, STK2 (NEK4), NKIAMRE,or HBO1 protein is expressed in a cell, and functional, e.g., physicaland chemical or phenotypic, changes are assayed to identify PKC-ζ,PLC-β1, FAK, FAK2, CK2, cMET, FEN1, REV1, APE1, CDK3, PIM1, CDC7L1,CDK7, CNK, PRL-3, STK2 (NEK4), NKIAMRE, or HBO1 and modulators ofcellular proliferation, e.g., tumor cell proliferation. Cells expressingPKC-ζ, PLC-β1, FAK, FAK2, CK2, cMET, FEN1, REV1, APE1, CDK3, PIM1,CDC7L1, CDK7, CNK, PRL-3, STK2 (NEK4), NKIAMRE, or HBO1 proteins canalso be used in binding assays and enzymatic assays. Any suitablefunctional effect can be measured, as described herein. For example,cellular morphology (e.g., cell volume, nuclear volume, cell perimeter,and nuclear perimeter), ligand binding, kinase activity, apoptosis, cellsurface marker expression, cellular proliferation, GFP positivity anddye dilution assays (e.g., cell tracker assays with dyes that bind tocell membranes), DNA synthesis assays (e.g., ³H-thymidine andfluorescent DNA-binding dyes such as BrdU or Hoescht dye with FACSanalysis), are all suitable assays to identify potential modulatorsusing a cell based system. Suitable cells for such cell based assaysinclude both primary cancer or tumor cells and cell lines, as describedherein, e.g., A549 (lung), MCF7 (breast, p53 wild-type), H1299 (lung,p53 null), Hela (cervical), PC3 (prostate, p53 mutant), MDA-MB-231(breast, p53 wild-type). Cancer cell lines can be p53 mutant, p53 null,or express wild type p53. The PKC-ζ, PLC-β1, FAK, FAK2, CK2, cMET, FEN1,REV1, APE1, CDK3, PIM1, CDC7L1, CDK7, CNK, PRL-3, STK2 (NEK4), NKIAMRE,or HBO1 protein can be naturally occurring or recombinant. Also,fragments of PKC-ζ, PLC-β1, FAK, FAK2, CK2, cMET, FEN1, REV1, APE1,CDK3, PIM1, CDC7L1, CDK7, CNK, PRL-3, STK2 (NEK4), NKIAMRE, or HBO1orchimeric PKC-ζ, PLC-β1, FAK, FAK2, CK2, cMET, FEN1, REV1, APE1, CDK3,PIM1, CDC7L1, CDK7, CNK, PRL-3, STK2 (NEK4), NKIAMRE, or HBO1 proteinswith enzymatic activity can be used in cell based assays.

[0208] Cellular PKC-ζ, PLC-β1, FAK, FAK2, CK2, cMET, FEN1, REV1, APE1,CDK3, PIM1, CDC7L1, CDK7, CNK, PRL-3, STK2 (NEK4), NKIAMRE, or HBO1polypeptide levels can be determined by measuring the level of proteinor mRNA. The level of PKC-ζ, PLC-β1, FAK, FAK2, CK2, cMET, FEN1, REV1,APE1, CDK3, PIM1, CDC7L1, CDK7, CNK, PRL-3, STK2 (NEK4), NKIAMRE, orHBO1 protein or proteins related to PKC-ζ, PLC-β1, FAK, FAK2, CK2, cMET,FEN1, REV1, APE1, CDK3, PIM1, CDC7L1, CDK7, CNK, PRL-3, STK2 (NEK4),NKIAMRE, or HBO1 are measured using immunoassays such as westernblotting, ELISA and the like with an antibody that selectively binds tothe PKC-ζ, PLC-β1, FAK, FAK2, CK2, cMET, FEN1, REV1, APE1, CDK3, PIM1,CDC7L1, CDK7, CNK, PRL-3, STK2 (NEK4), NKIAMRE, or HBO1 polypeptide or afragment thereof. For measurement of mRNA, amplification, e.g., usingPCR, LCR, or hybridization assays, e.g., northern hybridization, RNAseprotection, dot blotting, are preferred. The level of protein or mRNA isdetected using directly or indirectly labeled detection agents, e.g.,fluorescently or radioactively labeled nucleic acids, radioactively orenzymatically labeled antibodies, and the like, as described herein.

[0209] Alternatively, PKC-ζ, PLC-β1, FAK, FAK2, CK2, cMET, FEN1, REV1,APE1, CDK3, PIM1, CDC7L1, CDK7, CNK, PRL-3, STK2 (NEK4), NKIAMRE, orHBO1 expression can be measured using a reporter gene system. Such asystem can be devised using a PKC-ζ, PLC-β1, FAK, FAK2, CK2, cMET, FEN1,REV1, APE1, CDK3, PIM1, CDC7L1, CDK7, CNK, PRL-3, STK2 (NEK4), NKIAMRE,or HBO1 protein promoter operably linked to a reporter gene such aschloramphenicol acetyltransferase, firefly luciferase, bacterialluciferase, β-galactosidase and alkaline phosphatase. Furthermore, theprotein of interest can be used as an indirect reporter via attachmentto a second reporter such as red or green fluorescent protein (see,e.g., Mistili & Spector, Nature Biotechnology 15:961-964 (1997)). Thereporter construct is typically transfected into a cell. After treatmentwith a potential modulator, the amount of reporter gene transcription,translation, or activity is measured according to standard techniquesknown to those of skill in the art.

[0210] Animal Models

[0211] Animal models of cellular proliferation also find use inscreening for modulators of cellular proliferation. Similarly,transgenic animal technology including gene knockout technology, forexample as a result of homologous recombination with an appropriate genetargeting vector, or gene overexpression, will result in the absence orincreased expression of the PKC-ζ, PLC-β1, FAK, FAK2, CK2, cMET, FEN1,REV1, APE1, CDK3, PIM1, CDC7L1, CDK7, CNK, PRL-3, STK2 (NEK4), NKIAMRE,or HBO1. The same technology can also be applied to make knock-outcells. When desired, tissue-specific expression or knockout of thePKC-ζ, PLC-β1, FAK, FAK2, CK2, cMET, FEN1, REV1, APE1, CDK3, PIM1,CDC7L1, CDK7, CNK, PRL-3, STK2 (NEK4), NKIAMRE, or HBO1 protein may benecessary. Transgenic animals generated by such methods find use asanimal models of cellular proliferation and are additionally useful inscreening for modulators of cellular proliferation.

[0212] Knock-out cells and transgenic mice can be made by insertion of amarker gene or other heterologous gene into an endogenous PKC-ζ, PLC-β1,FAK, FAK2, CK2, cMET, FEN1, REV1, APE1, CDK3, PIM1, CDC7L1, CDK7, CNK,PRL-3, STK2 (NEK4), NKIAMRE, or HBO1 gene site in the mouse genome viahomologous recombination. Such mice can also be made by substituting anendogenous PKC-ζ, PLC-β1, FAK, FAK2, CK2, cMET, FEN1, REV1, APE1, CDK3,PIM1, CDC7L1, CDK7, CNK, PRL-3, STK2 (NEK4), NKIAMRE, or HBO1 with amutated version of the PKC-ζ, PLC-β1, FAK, FAK2, CK2, cMET, FEN1, REV1,APE1, CDK3, PIM1, CDC7L1, CDK7, CNK, PRL-3, STK2 (NEK4), NKIAMRE, orHBO1 gene, or by mutating an endogenous PKC-ζ, PLC-β1, FAK, FAK2, CK2,cMET, FEN1, REV1, APE1, CDK3, PIM1, CDC7L1, CDK7, CNK, PRL-3, STK2(NEK4), NKIAMRE, or HBO1, e.g., by exposure to carcinogens.

[0213] A DNA construct is introduced into the nuclei of embryonic stemcells. Cells containing the newly engineered genetic lesion are injectedinto a host mouse embryo, which is re-implanted into a recipient female.Some of these embryos develop into chimeric mice that possess germ cellspartially derived from the mutant cell line. Therefore, by breeding thechimeric mice it is possible to obtain a new line of mice containing theintroduced genetic lesion (see, e.g., Capecchi et al., Science 244:1288(1989)). Chimeric targeted mice can be derived according to Hogan etal., Manipulating the Mouse Embryo: A Laboratory Manual, Cold SpringHarbor Laboratory (1988) and Teratocarcinomas and Embryonic Stem Cells:A Practical Approach, Robertson, ed., IRL Press, Washington, D.C.,(1987).

[0214] Exemplary Assays

[0215] Enzymatic Activity Assays—In Vitro or Cell Based

[0216] In one embodiment, enzymatic assays using PKC-ζ, PLC-β1, FAK,FAK2, CK2, cMET, FEN1, REV1, APE1, CDK3, PIM1, CDC7L1, CDK7, CNK, PRL-3,STK2 (NEK4), NKIAMRE, or HBO1 can be used to identify modulators ofPKC-ζ, PLC-β1, FAK, FAK2, CK2, cMET, FEN1, REV1, APE1, CDK3, PIM1,CDC7L1, CDK7, CNK, PRL-3, STK2 (NEK4), NKIAMRE, or HBO1 activity, or toidentify proteins that bind to PKC-ζ, PLC-β1, FAK, FAK2, CK2, cMET,FEN1, REV1, APE1, CDK3, PIM1, CDC7L1, CDK7, CNK, PRL-3, STK2 (NEK4),NKIAMRE, or HBO1, e.g., PKC-ζ, PLC-β1, FAK, FAK2, CK2, cMET, FEN1, REV1,APE1, CDK3, PIM1, CDC7L1, CDK7, CNK, PRL-3, STK2 (NEK4), NKIAMRE, orHBO1 substrates. Full length wild type PKC-ζ, PLC-β1, FAK, FAK2, CK2,cMET, FEN1, REV1, APE1, CDK3, PIM1, CDC7L1, CDK7, CNK, PRL-3, STK2(NEK4), NKIAMRE, or HBO1, mutant PKC-ζ, PLC-β1, FAK, FAK2, CK2, cMET,FEN1, REV1, APE1, CDK3, PIM1, CDC7L1, CDK7, CNK, PRL-3, STK2 (NEK4),NKIAMRE, or HBO1, or the PKC-ζ, PLC-β1, FAK, FAK2, CK2, cMET, FEN1,REV1, APE1, CDK3, PIM1, CDC7L1, CDK7, CNK, PRL-3, STK2 (NEK4), NKIAMRE,or HBO1 enzymatic domain can be used in these assays. Such assays can beperformed in vitro, using recombinant PKC-ζ, PLC-β1, FAK, FAK2, CK2,cMET, FEN1, REV1, APE1, CDK3, PIM1, CDC7L1, CDK7, CNK, PRL-3, STK2(NEK4), NKIAMRE, or HBO1 or cellular lysates comprising endogenous orrecombinant PKC-ζ, PLC-β1, FAK, FAK2, CK2, cMET, FEN1, REV1, APE1, CDK3,PIM1, CDC7L1, CDK7, CNK, PRL-3, STK2 (NEK4), NKIAMRE, or HBO1, or can becell-based.

[0217] Soft Agar Growth or Colony Formation in Suspension

[0218] Normal cells require a solid substrate to attach and grow. Whenthe cells are transformed, they lose this phenotype and grow detachedfrom the substrate. For example, transformed cells can grow in stirredsuspension culture or suspended in semi-solid media, such as semi-solidor soft agar. The transformed cells, when transfected with tumorsuppressor genes, regenerate normal phenotype and require a solidsubstrate to attach and grow.

[0219] Soft agar growth or colony formation in suspension assays can beused to identify PKC-ζ, PLC-β1, FAK, FAK2, CK2, cMET, FEN1, REV1, APE1,CDK3, PIM1, CDC7L1, CDK7, CNK, PRL-3, STK2 (NEK4), NKIAMRE, or HBO1modulators. Typically, transformed host cells (e.g., cells that grow onsoft agar) are used in this assay. For example, RKO or HCT116 cell linescan be used. Techniques for soft agar growth or colony formation insuspension assays are described in Freshney, Culture of Animal Cells aManual of Basic Technique, 3^(rd) ed., Wiley-Liss, New York (1994),herein incorporated by reference. See also, the methods section ofGarkavtsev et al. (1996), supra, herein incorporated by reference.

[0220] Contact Inhibition and Density Limitation of Growth

[0221] Normal cells typically grow in a flat and organized pattern in apetri dish until they touch other cells. When the cells touch oneanother, they are contact inhibited and stop growing. When cells aretransformed, however, the cells are not contact inhibited and continueto grow to high densities in disorganized foci. Thus, the transformedcells grow to a higher saturation density than normal cells. This can bedetected morphologically by the formation of a disoriented monolayer ofcells or rounded cells in foci within the regular pattern of normalsurrounding cells. Alternatively, labeling index with [³H]-thymidine atsaturation density can be used to measure density limitation of growth.See Freshney (1994), supra. The transformed cells, when contacted withcellular proliferation modulators, regenerate a normal phenotype andbecome contact inhibited and would grow to a lower density.

[0222] Contact inhibition and density limitation of growth assays can beused to identify PKC-ζ, PLC-β1, FAK, FAK2, CK2, cMET, FEN1, REV1, APE1,CDK3, PIM1, CDC7L1, CDK7, CNK, PRL-3, STK2 (NEK4), NKIAMRE, or HBO1modulators which are capable of inhibiting abnormal proliferation andtransformation in host cells. Typically, transformed host cells (e.g.,cells that are not contact inhibited) are used in this assay. Forexample, RKO or HCT116 cell lines can be used. In this assay, labelingindex with [³H]-thymidine at saturation density is a preferred method ofmeasuring density limitation of growth. Transformed host cells arecontacted with a potential PKC-ζ, PLC-β1, FAK, FAK2, CK2, cMET, FEN1,REV1, APE1, CDK3, PIM1, CDC7L1, CDK7, CNK, PRL-3, STK2 (NEK4), NKIAMRE,or HBO1 modulator and are grown for 24 hours at saturation density innon-limiting medium conditions. The percentage of cells labeling with[³H]-thymidine is determined autoradiographically. See, Freshney (1994),supra. The host cells contacted with a PKC-ζ, PLC-β1, FAK, FAK2, CK2,cMET, FEN1, REV1, APE1, CDK3, PIM1, CDC7L1, CDK7, CNK, PRL-3, STK2(NEK4), NKIAMRE, or HBO1 modulator would give arise to a lower labelingindex compared to control (e.g., transformed host cells transfected witha vector lacking an insert).

[0223] Growth Factor or Serum Dependence

[0224] Growth factor or serum dependence can be used as an assay toidentify PKC-ζ, PLC-β1, FAK, FAK2, CK2, cMET, FEN1, REV1, APE1, CDK3,PIM1, CDC7L1, CDK7, CNK, PRL-3, STK2 (NEK4), NKIAMRE, or HBO1modulators. Transformed cells have a lower serum dependence than theirnormal counterparts (see, e.g., Temin, J. Natl. Cancer Insti. 37:167-175(1966); Eagle et al., J. Exp. Med. 131:836-879 (1970)); Freshney, supra.This is in part due to release of various growth factors by thetransformed cells. When transformed cells are contacted with a PKC-ζ,PLC-β1, FAK, FAK2, CK2, cMET, FEN1, REV1, APE1, CDK3, PIM1, CDC7L1,CDK7, CNK, PRL3, STK2 (NEK4), NKIAMRE, or HBO1 modulator, the cellswould reacquire serum dependence and would release growth factors at alower level.

[0225] Tumor Specific Markers Levels

[0226] Tumor cells release an increased amount of certain factors(hereinafter “tumor specific markers”) than their normal counterparts.For example, plasminogen activator (PA) is released from human glioma ata higher level than from normal brain cells (see, e.g., Gullino,Angiogenesis, tumor vascularization, and potential interference withtumor growth. In Mihich (ed.): “Biological Responses in Cancer.” NewYork, Academic Press, pp. 178-184 (1985)). Similarly, tumor angiogenesisfactor (TAF) is released at a higher level in tumor cells than theirnormal counterparts. See, e.g., Folkman, Angiogenesis and cancer, SemCancer Biol. (1992)).

[0227] Tumor specific markers can be assayed to identify PKC-ζ, PLC-β1,FAK, FAK2, CK2, cMET, FEN1, REV1, APE1, CDK3, PIM1, CDC7L1, CDK7, CNK,PRL-3, STK2 (NEK4), NKIAMRE, or HBO1 modulators which decrease the levelof release of these markers from host cells. Typically, transformed ortumorigenic host cells are used. Various techniques which measure therelease of these factors are described in Freshney (1994), supra. Also,see, Unkless et al., J. Biol. Chem. 249:4295-4305 (1974); Strickland &Beers, J. Biol. Chem. 251:5694-5702 (1976); Whur et al., Br. J. Cancer42:305-312 (1980); Gulino, Angiogenesis, tumor vascularization, andpotential interference with tumor growth. In Mihich, E. (ed):“Biological Responses in Cancer.” New York, Plenum (1985); FreshneyAnticancer Res. 5:111-130 (1985).

[0228] Invasiveness into Matrigel

[0229] The degree of invasiveness into Matrigel or some otherextracellular matrix constituent can be used as an assay to identifyPKC-ζ, PLC-β1, FAK, FAK2, CK2, cMET, FEN1, REV1, APE1, CDK3, PIM1,CDC7L1, CDK7, CNK, PRL-3, STK2 (NEK4), NKIAMRE, or HBO1 modulators whichare capable of inhibiting abnormal cell proliferation and tumor growth.Tumor cells exhibit a good correlation between malignancy andinvasiveness of cells into Matrigel or some other extracellular matrixconstituent. In this assay, tumorigenic cells are typically used as hostcells. Therefore, PKC-ζ, PLC-β1, FAK, FAK2, CK2, cMET, FEN1, REV1, APE1,CDK3, PIM1, CDC7L1, CDK7, CNK, PRL-3, STK2 (NEK4), NKIAMRE, or HBO1modulators can be identified by measuring changes in the level ofinvasiveness between the host cells before and after the introduction ofpotential modulators. If a compound modulates PKC-ζ, PLC-β1, FAK, FAK2,CK2, cMET, FEN1, REV1, APE1, CDK3, PIM1, CDC7L1, CDK7, CNK, PRL-3, STK2(NEK4), NKIAMRE, or HBO1, its expression in tumorigenic host cells wouldaffect invasiveness.

[0230] Techniques described in Freshney (1994), supra, can be used.Briefly, the level of invasion of host cells can be measured by usingfilters coated with Matrigel or some other extracellular matrixconstituent. Penetration into the gel, or through to the distal side ofthe filter, is rated as invasiveness, and rated histologically by numberof cells and distance moved, or by prelabeling the cells with ¹²⁵I andcounting the radioactivity on the distal side of the filter or bottom ofthe dish. See, e.g., Freshney (1984), supra.

[0231] Apoptosis Analysis

[0232] Apoptosis analysis can be used as an assay to identify PKC-ζ,PLC-β1, FAK, FAK2, CK2, cMET, FEN1, REV1, APE1, CDK3, PIM1, CDC7L1,CDK7, CNK, PRL-3, STK2 (NEK4), NKIAMRE, or HBO1 modulators. In thisassay, cell lines, such as RKO or HCT116, can be used to screen PKC-ζ,PLC-β1, FAK, FAK2, CK2, cMET, FEN1, REV1, APE1, CDK3, PIM1, CDC7L1,CDK7, CNK, PRL-3, STK2 (NEK4), NKIAMRE, or HBO1 modulators. Cells arecontacted with a putative PKC-ζ, PLC-β1, FAK, FAK2, CK2, cMET, FEN1,REV1, APE1, CDK3, PIM1, CDC7L1, CDK7, CNK, PRL-3, STK2 (NEK4), NKIAMRE,or HBO1 modulator. The cells can be co-transfected with a constructcomprising a marker gene, such as a gene that encodes green fluorescentprotein, or a cell tracker dye. The apoptotic change can be determinedusing methods known in the art, such as DAPI staining and TUNEL assayusing a fluorescent microscope. For TUNEL assay, commercially availablekit can be used (e.g., Fluorescein FragEL DNA Fragmentation DetectionKit (Oncogene Research Products, Cat.#QIA39)+Tetramethyl-rhodamine-5-dUTP (Roche, Cat. # 1534 378)). Cellscontacted with PKC-ζ, PLC-β1, FAK, FAK2, CK2, cMET, FEN1, REV1, APE1,CDK3, PIM1, CDC7L1, CDK7, CNK, PRL-3, STK2 (NEK4), NKIAMRE, or HBO1modulators would exhibit, e.g., an increased apoptosis compared tocontrol.

[0233] Cell Cycle Arrest Analysis

[0234] Cell cycle arrest can be used as an assay to identify PKC-ζ,PLC-β1, FAK, FAK2, CK2, cMET, FEN1, REV1, APE1, CDK3, PIM1, CDC7L1,CDK7, CNK, PRL-3, STK2 (NEK4), NKIAMRE, or HBO1 modulators. In thisassay, cell lines, such as RKO or HCT116, can be used to screen PKC-ζ,PLC-β1, FAK, FAK2, CK2, cMET, FEN1, REV1, APE1, CDK3, PIM1, CDC7L1,CDK7, CNK, PRL-3, STK2 (NEK4), NKIAMRE, or HBO1 modulators. The cellscan be co-transfected with a construct comprising a marker gene, such asa gene that encodes green fluorescent protein, or a cell tracker dye.Methods known in the art can be used to measure the degree of cell cyclearrest. For example, a propidium iodide signal can be used as a measurefor DNA content to determine cell cycle profiles on a flow cytometer.The percent of the cells in each cell cycle can be calculated. Cellscontacted with a PKC-ζ, PLC-β1, FAK, FAK2, CK2, cMET, FEN1, REV1, APE1,CDK3, PIM1, CDC7L1, CDK7, CNK, PRL-3, STK2 (NEK4), NKIAMRE, or HBO1modulator would exhibit, e.g., a higher number of cells that arearrested in G₁/G₀ phase, G₁/S phase, S/G₂ phase, G₂/M phase, or M/G₂phase compared to control.

[0235] Tumor Growth In Vivo

[0236] Effects of PKC-ζ, PLC-β1, FAK, FAK2, CK2, cMET, FEN1, REV1, APE1,CDK3, PIM1, CDC7L1, CDK7, CNK, PRL-3, STK2 (NEK4), NKIAMRE, or HBO1modulators on cell growth can be tested in transgenic orimmune-suppressed mice (e.g., xenograft models). Knock-out transgenicmice can be made, in which the endogenous PKC-ζ, PLC-β1, FAK, FAK2, CK2,cMET, FEN1, REV1, APE1, CDK3, PIM1, CDC7L1, CDK7, CNK, PRL-3, STK2(NEK4), NKIAMRE, or HBO1 gene is disrupted. Such knock-out mice can beused to study effects of PKC-ζ, PLC-β1, FAK, FAK2, CK2, cMET, FEN1,REV1, APE1, CDK3, PIM1, CDC7L1, CDK7, CNK, PRL-3, STK2 (NEK4), NKIAMRE,or HBO1, e.g., as a cancer model, as a means of assaying in vivo forcompounds that modulate PKC-ζ, PLC-β1, FAK, FAK2, CK2, cMET, FEN1, REV1,APE1, CDK3, PIM1, CDC7L1, CDK7, CNK, PRL-3, STK2 (NEK4), NKIAMRE, orHBO1, and to test the effects of restoring a wild-type or mutant PKC-ζ,PLC-β1, FAK, FAK2, CK2, cMET, FEN1, REV1, APE1, CDK3, PIM1, CDC7L1,CDK7, CNK, PRL-3, STK2 (NEK4), NKIAMRE, or HBO1 to a knock-out mice.

[0237] Knock-out cells and transgenic mice can be made by insertion of amarker gene or other heterologous gene into the endogenous PKC-ζ,PLC-β1, FAK, FAK2, CK2, cMET, FEN1, REV1, APE1, CDK3, PIM1, CDC7L1,CDK7, CNK, PRL-3, STK2 (NEK4), NKIAMRE, or HBO1 gene site in the mousegenome via homologous recombination. Such mice can also be made bysubstituting the endogenous PKC-ζ, PLC-β1, FAK, FAK2, CK2, cMET, FEN1,REV1, APE1, CDK3, PIM1, CDC7L1, CDK7, CNK, PRL-3, STK2 (NEK4), NKIAMRE,or HBO1 with a mutated version of PKC-ζ, PLC-β1, FAK, FAK2, CK2, cMET,FEN1, REV1, APE1, CDK3, PIM1, CDC7L1, CDK7, CNK, PRL-3, STK2 (NEK4),NKIAMRE, or HBO1, or by mutating the endogenous PKC-ζ, PLC-β1, FAK,FAK2, CK2, cMET, FEN1, REV1, APE1, CDK3, PIM1, CDC7L1, CDK7, CNK, PRL-3,STK2 (NEK4), NKIAMRE, or HBO1, e.g., by exposure to carcinogens.

[0238] A DNA construct is introduced into the nuclei of embryonic stemcells. Cells containing the newly engineered genetic lesion are injectedinto a host mouse embryo, which is re-implanted into a recipient female.Some of these embryos develop into chimeric mice that possess germ cellspartially derived from the mutant cell line. Therefore, by breeding thechimeric mice it is possible to obtain a new line of mice containing theintroduced genetic lesion (see, e.g., Capecchi et al., Science 244:1288(1989)). Chimeric targeted mice can be derived according to Hogan etal., Manipulating the Mouse Embryo: A Laboratory Manual, Cold SpringHarbor Laboratory (1988) and Teratocarcinomas and Embryonic Stem Cells:A Practical Approach, Robertson, ed., IRL Press, Washington, D.C.,(1987). These knock-out mice can be used as hosts to test the effects ofvarious PKC-ζ, PLC-β1, FAK, FAK2, CK2, cMET, FEN1, REV1, APE1, CDK3,PIM1, CDC7L1, CDK7, CNK, PRL-3, STK2 (NEK4), NKIAMRE, or HBO1 modulatorson cell growth.

[0239] Alternatively, various immune-suppressed or immune-deficient hostanimals can be used. For example, genetically athymic “nude” mouse (see,e.g., Giovanella et al., J. Natl. Cancer Inst. 52:921 (1974)), a SCIDmouse, a thymectomized mouse, or an irradiated mouse (see, e.g., Bradleyet al., Br. J. Cancer 38:263 (1978); Selby et al, Br. J. Cancer 41:52(1980)) can be used as a host for, e.g., xenografts. Transplantabletumor cells (typically about 10⁶ cells), such as, for example, humantumor cells, injected into isogenic hosts will produce invasive tumorsin a high proportions of cases, while normal cells of similar originwill not. Hosts are treated with PKC-ζ, PLC-β1, FAK, FAK2, CK2, cMET,FEN1, REV1, APE1, CDK3, PIM1, CDC7L1, CDK7, CNK, PRL-3, STK2 (NEK4),NKIAMRE, or HBO1 modulators, e.g., by injection. After a suitable lengthof time, preferably 4-8 weeks, tumor growth is measured (e.g., by volumeor by its two largest dimensions) and compared to the control. Tumorsthat have statistically significant reduction (using, e.g., Student's Ttest) are said to have inhibited growth. Using reduction of tumor sizeas an assay, PKC-ζ, PLC-β1, FAK, FAK2, CK2, cMET, FEN1, REV1, APE1,CDK3, PIM1, CDC7L1, CDK7, CNK, PRL-3, STK2 (NEK4), NKIAMRE, or HBO1modulators which are capable, e.g., of inhibiting abnormal cellproliferation can be identified.

[0240] B. Modulators

[0241] The compounds tested as modulators of PKC-ζ, PLC-β1, FAK, FAK2,CK2, cMET, FEN1, REV1, APE1, CDK3, PIM1, CDC7L1, CDK7, CNK, PRL-3, STK2(NEK4), NKIAMRE, or HBO1 protein can be any small organic molecule, or abiological entity, such as a protein, e.g., an antibody or peptide, asugar, a nucleic acid, e.g., an antisense oligonucleotide or a ribozyme,or a lipid. Alternatively, modulators can be genetically alteredversions of a PKC-ζ, PLC-β1, FAK, FAK2, CK2, cMET, FEN1, REV1, APE1,CDK3, PIM1, CDC7L1, CDK7, CNK, PRL-3, STK2 (NEK4), NKIAMRE, or HBO1protein. Typically, test compounds will be small organic molecules,peptides, circular peptides, RNAi, antisense molecules, ribozymes, andlipids.

[0242] Essentially any chemical compound can be used as a potentialmodulator or ligand in the assays of the invention, although most oftencompounds that can be dissolved in aqueous or organic (especiallyDMSO-based) solutions are used. The assays are designed to screen largechemical libraries by automating the assay steps and providing compoundsfrom any convenient source to assays, which are typically run inparallel (e.g., in microtiter formats on microtiter plates in roboticassays). It will be appreciated that there are many suppliers ofchemical compounds, including Sigma (St. Louis, Mo.), Aldrich (St.Louis, Mo.), Sigma-Aldrich (St. Louis, Mo.), Fluka Chemika-BiochemicaAnalytika (Buchs Switzerland) and the like.

[0243] In one preferred embodiment, high throughput screening methodsinvolve providing a combinatorial small organic molecule or peptidelibrary containing a large number of potential therapeutic compounds(potential modulator or ligand compounds). Such “combinatorial chemicallibraries” or “ligand libraries” are then screened in one or moreassays, as described herein, to identify those library members(particular chemical species or subclasses) that display a desiredcharacteristic activity. The compounds thus identified can serve asconventional “lead compounds” or can themselves be used as potential oractual therapeutics.

[0244] A combinatorial chemical library is a collection of diversechemical compounds generated by either chemical synthesis or biologicalsynthesis, by combining a number of chemical “building blocks” such asreagents. For example, a linear combinatorial chemical library such as apolypeptide library is formed by combining a set of chemical buildingblocks (amino acids) in every possible way for a given compound length(i.e., the number of amino acids in a polypeptide compound). Millions ofchemical compounds can be synthesized through such combinatorial mixingof chemical building blocks.

[0245] Preparation and screening of combinatorial chemical libraries iswell known to those of skill in the art. Such combinatorial chemicallibraries include, but are not limited to, peptide libraries (see, e.g.,U.S. Pat. No. 5,010,175, Furka, Int. J. Pept. Prot. Res. 37:487-493(1991) and Houghton et al., Nature 354:84-88 (1991)). Other chemistriesfor generating chemical diversity libraries can also be used. Suchchemistries include, but are not limited to: peptoids (e.g., PCTPublication No. WO 91/19735), encoded peptides (e.g., PCT PublicationNo. WO 93/20242), random bio-oligomers (e.g., PCT Publication No. WO92/00091), benzodiazepines (e.g., U.S. Pat. No. 5,288,514), diversomerssuch as hydantoins, benzodiazepines and dipeptides (Hobbs et al., Proc.Nat. Acad. Sci. USA 90:6909-6913 (19933)), vinylogous polypeptides(Hagihara et al., J. Amer. Chem. Soc. 114:6568 (1992)), nonpeptidalpeptidomimetics with glucose scaffolding (Hirschmann et al., J. Amer.Chem. Soc. 114:9217-9218 (1992)), analogous organic syntheses of smallcompound libraries (Chen et al., J. Amer. Chem. Soc. 116:2661 (1994)),oligocarbamates (Cho et al., Science 261:1303 (1993)), and/or peptidylphosphonates (Campbell et al., J. Org. Chem. 59:658 (1994)), nucleicacid libraries (see Ausubel, Berger and Sambrook, all supra), peptidenucleic acid libraries (see, e.g., U.S. Pat. No. 5,539,083), antibodylibraries (see, e.g., Vaughn et al., Nature Biotechnology, 14(3):309-314(1996) and PCT/US96/10287), carbohydrate libraries (see, e.g., Liang etal., Science, 274:1520-1522 (1996) and U.S. Pat. No. 5,593,853), smallorganic molecule libraries (see, e.g., benzodiazepines, Baum C&EN, Jan18, page 33 (1993); isoprenoids, U.S. Pat. No. 5,569,588;thiazolidinones and metathiazanojies, U.S. Pat. No. 5,549,974;pyrrolidines, U.S. Pat. Nos. 5,525,735 and 5,519,134; morpholinocompounds, U.S. Pat. No. 5,506,337; benzodiazepines, 5,288,514, and thelike).

[0246] Devices for the preparation of combinatorial libraries arecommercially available (see, e.g., 357 MPS, 390 MPS, Advanced Chem Tech,Louisville Ky., Symphony, Rainin, Woburn, Mass., 433A AppliedBiosystems, Foster City, Calif., 9050 Plus, Millipore, Bedford, Mass.).In addition, numerous combinatorial libraries are themselvescommercially available (see, e.g., ComGenex, Princeton, N.J.; Asinex,Moscow, RU; Tripos, Inc., St. Louis, Mo.; ChemStar, Ltd, Moscow, RU; 3DPharmaceuticals, Exton, Pa.; Martek Biosciences, Columbia, Md., etc.).

[0247] C. Solid State and Soluble High Throughput Assays

[0248] In one embodiment the invention provides soluble assays using aPKC-ζ, PLC-β1, FAK, FAK2, CK2, cMET, FEN1, REV1, APE1, CDK3, PIM1,CDC7L1, CDK7, CNK, PRL-3, STK2 (NEK4), NKIAMRE, or HBO1 protein, or acell or tissue expressing a PKC-ζ, PLC-β1, FAK, FAK2, CK2, cMET, FEN1,REV1, APE1, CDK3, PIM1, CDC7L1, CDK7, CNK, PRL-3, STK2 (NEK4), NKIAMRE,or HBO1 protein, either naturally occurring or recombinant. In anotherembodiment, the invention provides solid phase based in vitro assays ina high throughput format, where the PKC-ζ, PLC-β1, FAK, FAK2, CK2, cMET,FEN1, REV1, APE1, CDK3, PIM1, CDC7L1, CDK7, CNK, PRL-3, STK2 (NEK4),NKIAMRE, or HBO1 protein or PKC-ζ, PLC-β1, FAK, FAK2, CK2, cMET, FEN1,REV1, APE1, CDK3, PIM1, CDC7L1, CDK7, CNK, PRL-3, STK2 (NEK4), NKIAMRE,or HBO1 substrate is attached to a solid phase. Any one of the assaysdescribed herein can be adapted for high throughput screening.

[0249] In the high throughput assays of the invention, either soluble orsolid state, it is possible to screen up to several thousand differentmodulators or ligands in a single day. This methodology can be used forPKC-ζ, PLC-β1, FAK, FAK2, CK2, cMET, FEN1, REV1, APE1, CDK3, PIM1,CDC7L1, CDK7, CNK, PRL-3, STK2 (NEK4), NKIAMRE, or HBO1 proteins invitro, or for cell-based or membrane-based assays comprising a PKC-ζ,PLC-β1, FAK, FAK2, CK2, cMET, FEN1, REV1, APE1, CDK3, PIM1, CDC7L1,CDK7, CNK, PRL-3, STK2 (NEK4), NKIAMRE, or HBO1 protein. In particular,each well of a microtiter plate can be used to run a separate assayagainst a selected potential modulator, or, if concentration orincubation time effects are to be observed, every 5-10 wells can test asingle modulator. Thus, a single standard microtiter plate can assayabout 100 (e.g., 96) modulators. If 1536 well plates are used, then asingle plate can easily assay from about 100- about 1500 differentcompounds. It is possible to assay many plates per day; assay screensfor up to about 6,000, 20,000, 50,000, or more than 100,000 differentcompounds are possible using the integrated systems of the invention.

[0250] For a solid state reaction, the protein of interest or a fragmentthereof, e.g., an extracellular domain, or a cell or membrane comprisingthe protein of interest or a fragment thereof as part of a fusionprotein can be bound to the solid state component, directly orindirectly, via covalent or non covalent linkage. A tag for covalent ornon-covalent binding can be any of a variety of components. In general,a molecule which binds the tag (a tag binder) is fixed to a solidsupport, and the tagged molecule of interest is attached to the solidsupport by interaction of the tag and the tag binder.

[0251] A number of tags and tag binders can be used, based upon knownmolecular interactions well described in the literature. For example,where a tag has a natural binder, for example, biotin, protein A, orprotein G, it can be used in conjunction with appropriate tag binders(avidin, streptavidin, neutravidin, the Fc region of an immunoglobulin,etc.). Antibodies to molecules with natural binders such as biotin andappropriate tag binders are also widely available; see, SIGMAImmunochemicals 1998 catalogue SIGMA, St. Louis Mo.).

[0252] Similarly, any haptenic or antigenic compound can be used incombination with an appropriate antibody to form a tag/tag binder pair.Thousands of specific antibodies are commercially available and manyadditional antibodies are described in the literature. For example, inone common configuration, the tag is a first antibody and the tag binderis a second antibody which recognizes the first antibody. In addition toantibody-antigen interactions, receptor-ligand interactions are alsoappropriate as tag and tag-binder pairs. For example, agonists andantagonists of cell membrane receptors (e.g., cell receptor-ligandinteractions such as transferrin, c-kit, viral receptor ligands,cytokine receptors, chemokine receptors, interleukin receptors,immunoglobulin receptors and antibodies, the cadherein family, theintegrin family, the selectin family, and the like; see, e.g., Pigott &Power, The Adhesion Molecule Facts Book I (1993). Similarly, toxins andvenoms, viral epitopes, hormones (e.g., opiates, steroids, etc.),intracellular receptors (e.g. which mediate the effects of various smallligands, including steroids, thyroid hormone, retinoids and vitamin D;peptides), drugs, lectins, sugars, nucleic acids (both linear and cyclicpolymer configurations), oligosaccharides, proteins, phospholipids andantibodies can all interact with various cell receptors.

[0253] Synthetic polymers, such as polyurethanes, polyesters,polycarbonates, polyureas, polyamides, polyethyleneimines, polyarylenesulfides, polysiloxanes, polyimides, and polyacetates can also form anappropriate tag or tag binder. Many other tag/tag binder pairs are alsouseful in assay systems described herein, as would be apparent to one ofskill upon review of this disclosure.

[0254] Common linkers such as peptides, polyethers, and the like canalso serve as tags, and include polypeptide sequences, such as poly glysequences of between about 5 and 200 amino acids. Such flexible linkersare known to persons of skill in the art. For example, poly(ethelyneglycol) linkers are available from Shearwater Polymers, Inc. Huntsville,Ala. These linkers optionally have amide linkages, sulfhydryl linkages,or heterofunctional linkages.

[0255] Tag binders are fixed to solid substrates using any of a varietyof methods currently available. Solid substrates are commonlyderivatized or functionalized by exposing all or a portion of thesubstrate to a chemical reagent which fixes a chemical group to thesurface which is reactive with a portion of the tag binder. For example,groups which are suitable for attachment to a longer chain portion wouldinclude amines, hydroxyl, thiol, and carboxyl groups. Aminoalkylsilanesand hydroxyalkylsilanes can be used to functionalize a variety ofsurfaces, such as glass surfaces. The construction of such solid phasebiopolymer arrays is well described in the literature. See, e.g.,Merrifield, J. Am. Chem. Soc. 85:2149-2154 (1963) (describing solidphase synthesis of, e.g., peptides); Geysen et al., J. Immun. Meth.102:259-274 (1987) (describing synthesis of solid phase components onpins); Frank & Doring, Tetrahedron 44:60316040 (1988) (describingsynthesis of various peptide sequences on cellulose disks); Fodor etal., Science, 251:767-777 (1991); Sheldon et al., Clinical Chemistry39(4):718-719 (1993); and Kozal et al., Nature Medicine 2(7):753759(1996) (all describing arrays of biopolymers fixed to solid substrates).Non-chemical approaches for fixing tag binders to substrates includeother common methods, such as heat, cross-linking by UV radiation, andthe like.

[0256] Immunological Detection of PKC-R, PLC-β1, FAK, FAK2, CK2, cMET,FEN1, REV1, APE1, CDK3, PIM1, CDC7L1, CDK7, CNK, PR1;3, STK2 (NEK4),NKIAMRE, or HBO1 Polypeptides

[0257] In addition to the detection of PKC-ζ, PLC-β1, FAK, FAK2, CK2,cMET, FEN1, REV1, APE1, CDK3, PIM1, CDC7L1, CDK7, CNK, PRL-3, STK2(NEK4), NKIAMRE, or HBO1 gene and gene expression using nucleic acidhybridization technology, one can also use immunoassays to detect PKC-ζ,PLC-β1, FAK, FAK2, CK2, cMET, FEN1, REV1, APE1, CDK3, PIM1, CDC7L1,CDK7, CNK, PRL-3, STK2 (NEK4), NKIAMRE, or HBO1 proteins of theinvention. Such assays are useful for screening for modulators of PKC-ζ,PLC-β1, FAK, FAK2, CK2, cMET, FEN1, REV1, APE1, CDK3, PIM1, CDC7L1,CDK7, CNK, PRL-3, STK2 (NEK4), NKIAMRE, or HBO1, as well as fortherapeutic and diagnostic applications. Immunoassays can be used toqualitatively or quantitatively analyze PKC-ζ, PLC-β1, FAK, FAK2, CK2,cMET, FEN1, REV1, APE1, CDK3, PIM1, CDC7L1, CDK7, CNK, PRL-3, STK2(NEK4), NKIAMRE, or HBO1 protein. A general overview of the applicabletechnology can be found in Harlow & Lane, Antibodies: A LaboratoryManual (1988).

[0258] A. Production of Antibodies

[0259] Methods of producing polyclonal and monoclonal antibodies thatreact specifically with the PKC-ζ, PLC-β1, FAK, FAK2, CK2, cMET, FEN1,REV1, APE1, CDK3, PIM1, CDC7L1, CDK7, CNK, PRL-3, STK2 (NEK4), NKIAMRE,or HBO1 proteins are known to those of skill in the art (see, e.g.,Coligan, Current Protocols in Immunology (1991); Harlow & Lane, supra;Goding, Monoclonal Antibodies: Principles and Practice (2d ed. 1986);and Kohler & Milstein, Nature 256:495-497 (1975). Such techniquesinclude antibody preparation by selection of antibodies from librariesof recombinant antibodies in phage or similar vectors, as well aspreparation of polyclonal and monoclonal antibodies by immunizingrabbits or mice (see, e.g., Huse et al., Science 246:1275-1281 (1989);Ward et al., Nature 341:544-546 (1989)).

[0260] A number of immunogens comprising portions of PKC-ζ, PLC-β1, FAK,FAK2, CK2, cMET, FEN1, REV1, APE1, CDK3, PIM1, CDC7L1, CDK7, CNK, PRL-3,STK2 (NEK4), NKIAMRE, or HBO1 protein may be used to produce antibodiesspecifically reactive with PKC-ζ, PLC-β1, FAK, FAK2, CK2, cMET, FEN1,REV1, APE1, CDK3, PIM1, CDC7L1, CDK7, CNK, PRL-3, STK2 (NEK4), NKIAMRE,or HBO1 protein. For example, recombinant PKC-ζ, PLC-β1, FAK, FAK2, CK2,cMET, FEN1, REV1, APE1, CDK3, PIM1, CDC7L1, CDK7, CNK, PRL-3, STK2(NEK4), NKIAMRE, or HBO1 protein or an antigenic fragment thereof, canbe isolated as described herein. Recombinant protein can be expressed ineukaryotic or prokaryotic cells as described above, and purified asgenerally described above. Recombinant protein is the preferredimmunogen for the production of monoclonal or polyclonal antibodies.Alternatively, a synthetic peptide derived from the sequences disclosedherein and conjugated to a carrier protein can be used an immunogen.Naturally occurring protein may also be used either in pure or impureform. The product is then injected into an animal capable of producingantibodies. Either monoclonal or polyclonal antibodies may be generated,for subsequent use in immunoassays to measure the protein.

[0261] Methods of production of polyclonal antibodies are known to thoseof skill in the art. An inbred strain of mice (e.g., BALB/C mice) orrabbits is immunized with the protein using a standard adjuvant, such asFreund's adjuvant, and a standard immunization protocol. The animal'simmune response to the immunogen preparation is monitored by taking testbleeds and determining the titer of reactivity to the beta subunits.When appropriately high titers of antibody to the immunogen areobtained, blood is collected from the animal and antisera are prepared.Further fractionation of the antisera to enrich for antibodies reactiveto the protein can be done if desired (see, Harlow & Lane, supra).

[0262] Monoclonal antibodies may be obtained by various techniquesfamiliar to those skilled in the art. Briefly, spleen cells from ananimal immunized with a desired antigen are immortalized, commonly byfusion with a myeloma cell (see, Kohler & Milstein, Eur. J. Immunol.6:511-519 (1976)). Alternative methods of immortalization includetransformation with Epstein Barr Virus, oncogenes, or retroviruses, orother methods well known in the art. Colonies arising from singleimmortalized cells are screened for production of antibodies of thedesired specificity and affinity for the antigen, and yield of themonoclonal antibodies produced by such cells may be enhanced by varioustechniques, including injection into the peritoneal cavity of avertebrate host. Alternatively, one may isolate DNA sequences whichencode a monoclonal antibody or a binding fragment thereof by screeninga DNA library from human B cells according to the general protocoloutlined by Huse, et al., Science 246:1275-1281 (1989).

[0263] Monoclonal antibodies and polyclonal sera are collected andtitered against the immunogen protein in an immunoassay, for example, asolid phase immunoassay with the immunogen immobilized on a solidsupport. Typically, polyclonal antisera with a titer of 10⁴ or greaterare selected and tested for their cross reactivity against nou-PKC-ζ,PLC-β1, FAK, FAK2, CK2, cMET, FEN1, REV1, APE1, CDK3, PIM1, CDC7L1,CDK7, CNK, PRL-3, STK2 (NEK4), NKIAMRE, or HBO1 proteins, using acompetitive binding immunoassay. Specific polyclonal antisera andmonoclonal antibodies will usually bind with a K_(d) of at least about0.1 mM, more usually at least about 1 μM, preferably at least about 0.1μM or better, and most preferably, 0.01 μM or better. Antibodiesspecific only for a particular PKC-ζ, PLC-β1, FAK, FAK2, CK2, cMET,FEN1, REV1, APE1, CDK3, PIM1, CDC7L1, CDK7, CNK, PRL-3, STK2 (NEK4),NKIAMRE, or HBO1 ortholog, such as human PKC-ζ, PLC-β1, FAK, FAK2, CK2,cMET, FEN1, REV1, APE1, CDK3, PIM1, CDC7L1, CDK7, CNK, PRL-3, STK2(NEK4), NKIAMRE, or HBO1, can also be made, by subtracting out othercross-reacting orthologs from a species such as a non-human mammal. Inthis manner, antibodies that bind only to PKC-ζ, PLC-β1, FAK, FAK2, CK2,cMET, FEN1, REV1, APE1, CDK3, PIM1, CDC7L1, CDK7, CNK, PRL-3, STK2(NEK4), NKIAMRE, or HBO1 protein may be obtained.

[0264] Once the specific antibodies against PKC-ζ, PLC-β1, FAK, FAK2,CK2, cMET, FEN1, REV1, APE1, CDK3, PIM1, CDC7L1, CDK7, CNK, PRL-3, STK2(NEK4), NKIAMRE, or HBO1 protein are available, the protein can bedetected by a variety of immunoassay methods. In addition, the antibodycan be used therapeutically as a PKC-ζ, PLC-β1, FAK, FAK2, CK2, cMET,FEN1, REV1, APE1, CDK3, PIM1, CDC7L1, CDK7, CNK, PRL-3, STK2 (NEK4),NKIAMRE, or HBO1 modulators. For a review of immunological andimmunoassay procedures, see Basic and Clinical Immunology (Stites & Terreds., 7^(th) ed. 1991). Moreover, the immunoassays of the presentinvention can be performed in any of several configurations, which arereviewed extensively in Enzyme Immunoassay (Maggio, ed., 1980); andHarlow & Lane, supra.

[0265] B. Immunological Binding Assays

[0266] PKC-ζ, PLC-β1, FAK, FAK2, CK2, cMET, FEN1, REV1, APE1, CDK3,PIM1, CDC7L1, CDK7, CNK, PRL-3, STK2 (NEK4), NKIAMRE, or HBO1 proteincan be detected and/or quantified using any of a number of wellrecognized immunological binding assays (see, e.g., U.S. Pat. Nos.4,366,241; 4,376,110; 4,517,288; and 4,837,168). For a review of thegeneral immunoassays, see also Methods in Cell Biology: Antibodies inCell Biology, volume 37 (Asai, ed. 1993); Basic and Clinical Immunology(Stites & Terr, eds., 7th ed. 1991). Irmunological binding assays (orimmunoassays) typically use an antibody that specifically binds to aprotein or antigen of choice (in this case the PKC-ζ, PLC-β1, FAK, FAK2,CK2, cMET, FEN1, REV1, APE1, CDK3, PIM1, CDC7L1, CDK7, CNK, PRL-3, STK2(NEK4), NKIAMRE, or HBO1 protein or antigenic subsequence thereof). Theantibody (e.g., anti-PKC-ζ, PLC-β1, FAK, FAK2, CK2, cMET, FEN1, REV1,APE1, CDK3, PIM1, CDC7L1, CDK7, CNK, PRL-3, STK2 (NEK4), NKIAMRE, orHBO1) may be produced by any of a number of means well known to those ofskill in the art and as described above.

[0267] Immunoassays also often use a labeling agent to specifically bindto and label the complex formed by the antibody and antigen. Thelabeling agent may itself be one of the moieties comprising theantibody/antigen complex. Thus, the labeling agent may be a labeledPKC-ζ, PLC-β1, FAK, FAK2, CK2, cMET, FEN1, REV1, APE1, CDK3, PIM1,CDC7L1, CDK7, CNK, PRL-3, STK2 (NEK4), NKIAMRE, or HBO1 or a labeledanti-PKC-ζ, PLC-β1, FAK, FAK2, CK2, cMET, FEN1, REV1, APE1, CDK3, PIM1,CDC7L1, CDK7, CNK, PRL-3, STK2 (NEK4), NKIAMRE, or HBO1 antibody.Alternatively, the labeling agent may be a third moiety, such asecondary antibody, that specifically binds to the antibody/PKC-ζ,PLC-β1, FAK, FAK2, CK2, cMET, FEN1, REV1, APE1, CDK3, PIM1, CDC7L1,CDK7, CNK, PRL-3, STK2 (NEK4), NKIAMRE, or HBO1 complex (a secondaryantibody is typically specific to antibodies of the species from whichthe first antibody is derived). Other proteins capable of specificallybinding immunoglobulin constant regions, such as protein A or protein Gmay also be used as the label agent. These proteins exhibit a strongnon-immunogenic reactivity with immunoglobulin constant regions from avariety of species (see, e.g., Kronval et al., J. Immunol. 111:1401-1406(1973); Akerstrom et al., J. Immunol. 135:2589-2542 (1985)). Thelabeling agent can be modified with a detectable moiety, such as biotin,to which another molecule can specifically bind, such as streptavidin. Avariety of detectable moieties are well known to those skilled in theart.

[0268] Throughout the assays, incubation and/or washing steps may berequired after each combination of reagents. Incubation steps can varyfrom about 5 seconds to several hours, optionally from about 5 minutesto about 24 hours. However, the incubation time will depend upon theassay format, antigen, volume of solution, concentrations, and the like.Usually, the assays will be carried out at ambient temperature, althoughthey can be conducted over a range of temperatures, such as 10° C. to40° C.

[0269] Non-Competitive Assay Formats

[0270] Immunoassays for detecting PKC-ζ, PLC-β1, FAK, FAK2, CK2, cMET,FEN1, REV1, APE1, CDK3, PIM1, CDC7L1, CDK7, CNK, PRL-3, STK2 (NEK4),NKIAMRE, or HBO1 in samples may be either competitive or noncompetitive.Noncompetitive immunoassays are assays in which the amount of antigen isdirectly measured. In one preferred “sandwich” assay, for example, theanti-PKC-ζ, PLC-β1, FAK, FAK2, CK2, cMET, FEN1, REV1, APE1, CDK3, PIM1,CDC7L1, CDK7, CNK, PRL-3, STK2 (NEK4), NKIAMRE, or HBO1 antibodies canbe bound directly to a solid substrate on which they are immobilized.These immobilized antibodies then capture PKC-ζ, PLC-β1, FAK, FAK2, CK2,cMET, FEN1, REV1, APE1, CDK3, PIM1, CDC7L1, CDK7, CNK, PRL-3, STK2(NEK4), NKIAMRE, or HBO1 present in the test sample. PKC-ζ, PLC-β1, FAK,FAK2, CK2, cMET, FEN1, REV1, APE1, CDK3, PIM1, CDC7L1, CDK7, CNK, PRL-3,STK2 (NEK4), NKIAMRE, or HBO1 proteins thus immobilized are then boundby a labeling agent, such as a second PKC-ζ, PLC-β1, FAK, FAK2, CK2,cMET, FEN1, REV1, APE1, CDK3, PIM1, CDC7L1, CDK7, CNK, PRL-3, STK2(NEK4), NKIAMRE, or HBO1 antibody bearing a label. Alternatively, thesecond antibody may lack a label, but it may, in turn, be bound by alabeled third antibody specific to antibodies of the species from whichthe second antibody is derived. The second or third antibody istypically modified with a detectable moiety, such as biotin, to whichanother molecule specifically binds, e.g., streptavidin, to provide adetectable moiety.

[0271] Competitive Assay Formats

[0272] In competitive assays, the amount of PKC-ζ, PLC-β1, FAK, FAK2,CK2, cMET, FEN1, REV1, APE1, CDK3, PIM1, CDC7L1, CDK7, CNK, PRL-3, STK2(NEK4), NKIAMRE, or HBO1 protein present in the sample is measuredindirectly by measuring the amount of a known, added (exogenous) PKC-ζ,PLC-β1, FAK, FAK2, CK2, cMET, FEN1, REV1, APE1, CDK3, PIM1, CDC7L1,CDK7, CNK, PRL-3, STK2 (NEK4), NKIAMRE, or HBO1 protein displaced(competed away) from an anti-PKC-ζ, PLC-β1, FAK, FAK2, CK2, cMET, FEN1,REV1, APE1, CDK3, PIM1, CDC7L1, CDK7, CNK, PRL-3, STK2 (NEK4), NKIAMRE,or HBO1 antibody by the unknown PKC-ζ, PLC-β1, FAK, FAK2, CK2, cMET,FEN1, REV1, APE1, CDK3, PIM1, CDC7L1, CDK7, CNK, PRL-3, STK2 (NEK4),NKIAMRE, or HBO1 protein present in a sample. In one competitive assay,a known amount of PKC-ζ, PLC-β1, FAK, FAK2, CK2, cMET, FEN1, REV1, APE1,CDK3, PIM1, CDC7L1, CDK7, CNK, PRL-3, STK2 (NEK4), NKIAMRE, or HBO1protein is added to a sample and the sample is then contacted with anantibody that specifically binds to PKC-ζ, PLC-β1, FAK, FAK2, CK2, cMET,FEN1, REV1, APE1, CDK3, PIM1, CDC7L1, CDK7, CNK, PRL-3, STK2 (NEK4),NKIAMRE, or HBO1 protein. The amount of exogenous PKC-ζ, PLC-β1, FAK,FAK2, CK2, cMET, FEN1, REV1, APE1, CDK3, PIM1, CDC7L1, CDK7, CNK, PRL-3,STK2 (NEK4), NKIAMRE, or HBO1 protein bound to the antibody is inverselyproportional to the concentration of PKC-ζ, PLC-β1, FAK, FAK2, CK2,cMET, FEN1, REV1, APE1, CDK3, PIM1, CDC7L1, CDK7, CNK, PRL-3, STK2(NEK4), NKIAMRE, or HBO1 protein present in the sample. In aparticularly preferred embodiment, the antibody is immobilized on asolid substrate. The amount of PKC-ζ, PLC-β1, FAK, FAK2, CK2, cMET,FEN1, REV1, APE1, CDK3, PIM1, CDC7L1, CDK7, CNK, PRL-3, STK2 (NEK4),NKIAMRE, or HBO1 protein bound to the antibody may be determined eitherby measuring the amount of PKC-ζ, PLC-β1, FAK, FAK2, CK2, cMET, FEN1,REV1, APE1, CDK3, PIM1, CDC7L1, CDK7, CNK, PRL-3, STK2 (NEK4), NKIAMRE,or HBO1 present in PKC-ζ, PLC-β1, FAK, FAK2, CK2, cMET, FEN1, REV1,APE1, CDK3, PIM1, CDC7L1, CDK7, CNK, PRL-3, STK2 (NEK4), NKIAMRE, orHBO1 protein/antibody complex, or alternatively by measuring the amountof remaining uncomplexed protein. The amount of PKC-ζ, PLC-β1, FAK,FAK2, CK2, cMET, FEN1, REV1, APE1, CDK3, PIM1, CDC7L1, CDK7, CNK, PRL-3,STK2 (NEK4), NKIAMRE, or HBO1 protein may be detected by providing alabeled PKC-ζ, PLC-β1, FAK, FAK2, CK2, cMET, FEN1, REV1, APE1, CDK3,PIM1, CDC7L1, CDK7, CNK, PRL-3, STK2 (NEK4), NKIAMRE, or HBO1 molecule.

[0273] A hapten inhibition assay is another preferred competitive assay.In this assay the known PKC-ζ, PLC-β1, FAK, FAK2, CK2, cMET, FEN1, REV1,APE1, CDK3, PIM1, CDC7L1, CDK7, CNK., PRL-3, STK2 (NEK4), NKIAMRE, orHBO1 protein is immobilized on a solid substrate. A known amount ofanti-PKC-ζ, PLC-β1, FAK, FAK2, CK2, cMET, FEN1, REV1, APE1, CDK3, PIM1,CDC7L1, CDK7, CNK, PRL-3, STK2 (NEK4), NKIAMRE, or HBO1 antibody isadded to the sample, and the sample is then contacted with theimmobilized PKC-ζ, PLC-β1, FAK, FAK2, CK2, cMET, FEN1, REV1, APE1, CDK3,PIM1, CDC7L1, CDK7, CNK, PRL-3, STK2 (NEK4), NKIAMRE, or HBO1. Theamount of anti-PKC-ζ, PLC-β61, FAK, FAK2, CK2, cMET, FEN1, REV1, APE1,CDK3, PIM1, CDC7L1, CDK7, CNK, PRL-3, STK2 (NEK4), NKIAMRE, or HBO1antibody bound to the known immobilized PKC-ζ, PLC-β1, FAK, FAK2, CK2,cMET, FEN1, REV1, APE1, CDK3, PIM1, CDC7L1, CDK7, CNK, PRL-3, STK2(NEK4), NKIAMRE, or HBO1 is inversely proportional to the amount ofPKC-ζ, PLC-β1, FAK, FAK2, CK2, cMET, FEN1, REV1, APE1, CDK3, PIM1,CDC7L1, CDK7, CNK, PRL-3, STK2 (NEK4), NKIAMRE, or HBO1 protein presentin the sample. Again, the amount of immobilized antibody may be detectedby detecting either the immobilized fraction of antibody or the fractionof the antibody that remains in solution. Detection may be direct wherethe antibody is labeled or indirect by the subsequent addition of alabeled moiety that specifically binds to the antibody as describedabove.

[0274] Cross-Reactivity Determinations

[0275] Immunoassays in the competitive binding format can also be usedfor crossreactivity determinations. For example, a PKC-ζ, PLC-β1, FAK,FAK2, CK2, cMET, FEN1, REV1, APE1, CDK3, PIM1, CDC7L1, CDK7, CNK, PRL-3,STK2 (NEK4), NKIAMRE, or HBO1 can be immobilized to a solid support.Proteins (e.g., PKC-ζ, PLC-β1, FAK, FAK2, CK2, cMET, FEN1, REV1, APE1,CDK3, PIM1, CDC7L1, CDK7, CNK, PRL-3, STK2 (NEK4), NKIAMRE, or HBO1 andhomologs) are added to the assay that compete for binding of theantisera to the immobilized antigen. The ability of the added proteinsto compete for binding of the antisera to the immobilized protein iscompared to the ability of the PKC-ζ, PLC-β1, FAK, FAK2, CK2, cMET,FEN1, REV1, APE1, CDK3, PIM1, CDC7L1, CDK7, CNK, PRL-3, STK2 (NEK4),NKIAMRE, or HBO1 protein to compete with itself. The percentcrossreactivity for the above proteins is calculated, using standardcalculations. Those antisera with less than 10% crossreactivity witheach of the added proteins listed above are selected and pooled. Thecross-reacting antibodies are optionally removed from the pooledantisera by immunoabsorption with the added considered proteins, e.g.,distantly related homologs.

[0276] The immunoabsorbed and pooled antisera are then used in acompetitive binding immunoassay as described above to compare a secondprotein, thought to be perhaps an allele or polymorphic variant of aPKC-ζ, PLC-β1, FAK, FAK2, CK2, cMET, FEN1, REV1, APE1, CDK3, PIM1,CDC7L1, CDK7, CNK, PRL-3, STK2 (NEK4), NKIAMRE, or HBO1 protein, to theimmunogen protein. In order to make this comparison, the two proteinsare each assayed at a wide range of concentrations and the amount ofeach protein required to inhibit 50% of the binding of the antisera tothe immobilized protein is determined. If the amount of the secondprotein required to inhibit 50% of binding is less than 10 times theamount of the PKC-ζ, PLC-β1, FAK, FAK2, CK2, cMET, FEN1, REV1, APE1,CDK3, PIM1, CDC7L1, CDK7, CNK, PRL-3, STK2 (NEK4), NKIAMRE, or HBO1protein that is required to inhibit 50% of binding, then the secondprotein is said to specifically bind to the polyclonal antibodiesgenerated to PKC-ζ, PLC-β1, FAK, FAK2, CK2, cMET, FEN1, REV1, APE1,CDK3, PIM1, CDC7L1, CDK7, CNK, PRL-3, STK2 (NEK4), NKIAMRE, or HBO1immunogen.

[0277] Other Assay Formats

[0278] Western blot (immunoblot) analysis is used to detect and quantifythe presence of PKC-ζ, PLC-β1, FAK, FAK2, CK2, cMET, FEN1, REV1, APE1,CDK3, PIM1, CDC7L1, CDK7, CNK, PRL-3, STK2 (NEK4), NKIAMRE, or HBO1 inthe sample. The technique generally comprises separating sample proteinsby gel electrophoresis on the basis of molecular weight, transferringthe separated proteins to a suitable solid support, (such as anitrocellulose filter, a nylon filter, or derivatized nylon filter), andincubating the sample with the antibodies that specifically bind PKC-ζ,PLC-β1, FAK, FAK2, CK2, cMET, FEN1, REV1, APE1, CDK3, PIM1, CDC7L1,CDK7, CNK, PRL-3, STK2 (NEK4), NKIAMRE, or HBO1. The anti-PKC-ζ, PLC-β1,FAK, FAK2, CK2, cMET, FEN1, REV1, APE1, CDK3, PIM1, CDC7L1, CDK7, CNK,PRL-3, STK2 (NEK4), NKIAMRE, or HBO1 antibodies specifically bind to thePKC-ζ, PLC-β1, FAK, FAK2, CK2, cMET, FEN1, REV1, APE1, CDK3, PIM1,CDC7L1, CDK7, CNK, PRL-3, STK2 (NEK4), NKIAMRE, or HBO1 on the solidsupport. These antibodies may be directly labeled or alternatively maybe subsequently detected using labeled antibodies (e.g., labeled sheepanti-mouse antibodies) that specifically bind to the anti-PKC-ζ, PLC-β1,FAK, FAK2, CK2, cMET, FEN1, REV1, APE1, CDK3, PIM1, CDC7L1, CDK7, CNK,PRL-3, STK2 (NEK4), NKIAMRE, or HBO1 antibodies.

[0279] Other assay formats include liposome immunoassays (LIA), whichuse liposomes designed to bind specific molecules (e.g., antibodies) andrelease encapsulated reagents or markers. The released chemicals arethen detected according to standard techniques (see Monroe et al., Amer.Clin. Prod. Rev. 5:34-41 (1986)).

[0280] Reduction of Non-Specific Binding

[0281] One of skill in the art will appreciate that it is oftendesirable to minimize non-specific binding in immunoassays.Particularly, where the assay involves an antigen or antibodyimmobilized on a solid substrate it is desirable to minimize the amountof non-specific binding to the substrate. Means of reducing suchnon-specific binding are well known to those of skill in the art.Typically, this technique involves coating the substrate with aproteinaceous composition. In particular, protein compositions such asbovine serum albumin (BSA), nonfat powdered milk, and gelatin are widelyused with powdered milk being most preferred.

[0282] Labels

[0283] The particular label or detectable group used in the assay is nota critical aspect of the invention, as long as it does not significantlyinterfere with the specific binding of the antibody used in the assay.The detectable group can be any material having a detectable physical orchemical property. Such detectable labels have been well-developed inthe field of immunoassays and, in general, most any label useful in suchmethods can be applied to the present invention. Thus, a label is anycomposition detectable by spectroscopic, photochemical, biochemical,immunochemical, electrical, optical or chemical means. Useful labels inthe present invention include magnetic beads (e.g., DYNABEADS™),fluorescent dyes (e.g., fluorescein isothiocyanate, Texas red,rhodamine, and the like), radiolabels (e.g., ³H, ¹²⁵I, ³⁵S, ¹⁴C, or³²P), enzymes (e.g., horse radish peroxidase, alkaline phosphatase andothers commonly used in an ELISA), and calorimetric labels such ascolloidal gold or colored glass or plastic beads (e.g., polystyrene,polypropylene, latex, etc.).

[0284] The label may be coupled directly or indirectly to the desiredcomponent of the assay according to methods well known in the art. Asindicated above, a wide variety of labels may be used, with the choiceof label depending on sensitivity required, ease of conjugation with thecompound, stability requirements, available instrumentation, anddisposal provisions.

[0285] Non-radioactive labels are often attached by indirect means.Generally, a ligand molecule (e.g., biotin) is covalently bound to themolecule. The ligand then binds to another molecules (e.g.,streptavidin) molecule, which is either inherently detectable orcovalently bound to a signal system, such as a detectable enzyme, afluorescent compound, or a chemiluminescent compound. The ligands andtheir targets can be used in any suitable combination with antibodiesthat recognize PKC-ζ, PLC-β1, FAK, FAK2, CK2, cMET, FEN1, REV1, APE1,CDK3, PIM1, CDC7L1, CDK7, CNK, PRL-3, STK2 (NEK4), NKIAMRE, or HBO1protein, or secondary antibodies that recognize anti-PKC-ζ, PLC-β1, FAK,FAK2, CK2, cMET, FEN1, REV1, APE1, CDK3, PIM1, CDC7L1, CDK7, CNK, PRL-3,STK2 (NEK4), NKIAMRE, or HBO1.

[0286] The molecules can also be conjugated directly to signalgenerating compounds, e.g., by conjugation with an enzyme orfluorophore. Enzymes of interest as labels will primarily be hydrolases,particularly phosphatases, esterases and glycosidases, or oxidotases,particularly peroxidases. Fluorescent compounds include fluorescein andits derivatives, rhodamine and its derivatives, dansyl, umbelliferone,etc. Chemiluminescent compounds include luciferin, and2,3-dihydrophthalazinediones, e.g., luminol. For a review of variouslabeling or signal producing systems that may be used, see U.S. Pat. No.4,391,904.

[0287] Means of detecting labels are well known to those of skill in theart. Thus, for example, where the label is a radioactive label, meansfor detection include a scintillation counter or photographic film as inautoradiography. Where the label is a fluorescent label, it may bedetected by exciting the fluorochrome with the appropriate wavelength oflight and detecting the resulting fluorescence. The fluorescence may bedetected visually, by the use of electronic detectors such as chargecoupled devices (CCDs) or photomultipliers and the like. Similarly,enzymatic labels may be detected by providing the appropriate substratesfor the enzyme and detecting the resulting reaction product.Colorimetric or chemiluminescent labels may be detected simply byobserving the color associated with the label. Thus, in various dipstickassays, conjugated gold often appears pink, while various conjugatedbeads appear the color of the bead.

[0288] Some assay formats do not require the use of labeled components.For instance, agglutination assays can be used to detect the presence ofthe target antibodies. In this case, antigen-coated particles areagglutinated by samples comprising the target antibodies. In thisformat, none of the components need be labeled and the presence of thetarget antibody is detected by simple visual inspection.

[0289] Cellular Transfection and Gene Therapy

[0290] The present invention provides the nucleic acids of PKC-ζ,PLC-β1, FAK, FAK2, CK2, cMET, FEN1, REV1, APE1, CDK3, PIM1, CDC7L1,CDK7, CNK, PRL-3, STK2 (NEK43), NKIAMRE, or HBO1 protein for thetransfection of cells in vitro and in vivo. These nucleic acids can beinserted into any of a number of well-known vectors for the transfectionof target cells and organisms as described below. The nucleic acids aretransfected into cells, ex vivo or in vivo, through the interaction ofthe vector and the target cell. The nucleic acid, under the control of apromoter, then expresses a PKC-ζ, PLC-β1, FAK, FAK2, CK2, cMET, FEN1,REV1, APE1, CDK3, PIM1, CDC7L1, CDK7, CNK, PRL-3, STK2 (NEK4), NKIAMRE,or HBO1 protein of the present invention, thereby mitigating the effectsof absent, partial inactivation, or abnormal expression of a PKC-ζ,PLC-β1, FAK, FAK2, CK2, cMET, FEN1, REV1, APE1, CDK3, PIM1, CDC7L1,CDK7, CNK, PRL-3, STK2 (NEK4), NKIAMRE, or HBO1 gene, particularly as itrelates to cellular proliferation. The compositions are administered toa patient in an amount sufficient to elicit a therapeutic response inthe patient. An amount adequate to accomplish this is defined as“therapeutically effective dose or amount.”

[0291] Such gene therapy procedures have been used to correct acquiredand inherited genetic defects, cancer, and other diseases in a number ofcontexts. The ability to express artificial genes in humans facilitatesthe prevention and/or cure of many important human diseases, includingmany diseases which are not amenable to treatment by other therapies(for a review of gene therapy procedures, see Anderson, Science256:808-813 (1992); Nabel & Felgner, TIBTECH 11:211-217 (1993); Mitani &Caskey, TIBTECH 11:162-166 (1993); Mulligan, Science 926-932 (1993);Dillon, TIBTECH 11:167-175 (1993); Miller, Nature 357:455-460 (1992);Van Brunt, Biotechnology 6(10):1149-1154 (1998); Vigne, RestorativeNeurology and Neuroscience 8:35-36 (1995); Kremer & Perricaudet, BritishMedical Bulletin 51(1):31-44 (1995); Haddada et al., in Current Topicsin Microbiology and Immunology (Doerfler & Böhn eds., 1995); and Yu etal., Gene Therapy 1:13-26 (1994)).

[0292] Pharmaceutical Compositions and Administration

[0293] Pharmaceutically acceptable carriers are determined in part bythe particular composition being administered (e.g., nucleic acid,protein, modulatory compounds or transduced cell), as well as by theparticular method used to administer the composition. Accordingly, thereare a wide variety of suitable formulations of pharmaceuticalcompositions of the present invention (see, e.g., Remington'sPharmaceutical Sciences, 17^(th) ed., 1989). Administration can be inany convenient manner, e.g., by injection, oral administration,inhalation, transdermal application, or rectal administration.

[0294] Formulations suitable for oral administration can consist of (a)liquid solutions, such as an effective amount of the packaged nucleicacid suspended in diluents, such as water, saline or PEG 400; (b)capsules, sachets or tablets, each containing a predetermined amount ofthe active ingredient, as liquids, solids, granules or gelatin; (c)suspensions in an appropriate liquid; and (d) suitable emulsions. Tabletforms can include one or more of lactose, sucrose, mannitol, sorbitol,calcium phosphates, corn starch, potato starch, microcrystallinecellulose, gelatin, colloidal silicon dioxide, talc, magnesium stearate,stearic acid, and other excipients, colorants, fillers, binders,diluents, buffering agents, moistening agents, preservatives, flavoringagents, dyes, disintegrating agents, and pharmaceutically compatiblecarriers. Lozenge forms can comprise the active ingredient in a flavor,e.g., sucrose, as well as pastilles comprising the active ingredient inan inert base, such as gelatin and glycerin or sucrose and acaciaemulsions, gels, and the like containing, in addition to the activeingredient, carriers known in the art.

[0295] The compound of choice, alone or in combination with othersuitable components, can be made into aerosol formulations (i.e., theycan be “nebulized”) to be administered via inhalation. Aerosolformulations can be placed into pressurized acceptable propellants, suchas dichlorodifluoromethane, propane, nitrogen, and the like.

[0296] Formulations suitable for parenteral administration, such as, forexample, by intraarticular (in the joints), intravenous, intramuscular,intradermal, intraperitoneal, and subcutaneous routes, include aqueousand non-aqueous, isotonic sterile injection solutions, which can containantioxidants, buffers, bacteriostats, and solutes that render theformulation isotonic with the blood of the intended recipient, andaqueous and non-aqueous sterile suspensions that can include suspendingagents, solubilizers, thickening agents, stabilizers, and preservatives.In the practice of this invention, compositions can be administered, forexample, by intravenous infusion, orally, topically, intraperitoneally,intravesically or intrathecally. Parenteral administration andintravenous administration are the preferred methods of administration.The formulations of commends can be presented in unit-dose or multi-dosesealed containers, such as ampules and vials.

[0297] Injection solutions and suspensions can be prepared from sterilepowders, granules, and tablets of the kind previously described. Cellstransduced by nucleic acids for ex vivo therapy can also be administeredintravenously or parenterally as described above.

[0298] The dose administered to a patient, in the context of the presentinvention should be sufficient to effect a beneficial therapeuticresponse in the patient over time. The dose will be determined by theefficacy of the particular vector employed and the condition of thepatient, as well as the body weight or surface area of the patient to betreated. The size of the dose also will be determined by the existence,nature, and extent of any adverse side-effects that accompany theadministration of a particular vector, or transduced cell type in aparticular patient.

[0299] In determining the effective amount of the vector to beadministered in the treatment or prophylaxis of conditions owing todiminished or aberrant expression of the PKC-ζ, PLC-β1, FAK, FAK2, CK2,cMET, FEN1, REV1, APE1, CDK3, PIM1, CDC7L1, CDK7, CNK, PRL-3, STK2(NEK4), NKIAMRE, or HBO1 protein, the physician evaluates circulatingplasma levels of the vector, vector toxicities, progression of thedisease, and the production of anti-vector antibodies. In general, thedose equivalent of a naked nucleic acid from a vector is from about 1 μgto 100 μg for a typical 70 kilogram patient, and doses of vectors whichinclude a retroviral particle are calculated to yield an equivalentamount of therapeutic nucleic acid.

[0300] For administration, compounds and transduced cells of the presentinvention can be administered at a rate determined by the LD-50 of theinhibitor, vector, or transduced cell type, and the side-effects of theinhibitor, vector or cell type at various concentrations, as applied tothe mass and overall health of the patient. Administration can beaccomplished via single or divided doses.

EXAMPLES

[0301] The following examples are offered to illustrate, but not tolimit the claimed invention.

Example 1 Identification of Genes That Modulate Cell Proliferation UsingImmunoprecipitation Assays

[0302] PKCζ, PLCβ1, cMET, PIM1, and NKIAMRE were identified asmodulators of cell proliferation using co-immunoprecipitation assaysknown to those of skill in the art (see, e.g., Harlow and Lane, supra).More specifically, PKCζ, PLCβ1, cMET, PIM1, and NKIAMREco-immunoprecipitated with cell cycle modulating proteins previouslybound to a monoclonal antibody and thus were identified as modulators ofcell proliferation. In particular, PKCζ was identified using themonoclonal antibody ATM (specific for a nucleophosphoprotein involved inataxia telangiectasia); PLCβ1 was identified using the monoclonalantibody p48 (specific for a subunit of the RB tumor suppressor gene);cMET was identified using the monoclonal antibody RbAp48 (specific for afusion protein corresponding to amino acids 1-425 of human RbAp48); PIM1was identified using the monoclonal antibody p21 (specific for the tumorsuppressor gene p21); and NKIAMRE was identified using the monoclonalantibody RbAp48.

Example 2 Identification of Genes That Modulate Cell Proliferation UsingYeast Two Hybrid Assays

[0303] FAK, FAK2, CK2, FEN2, REV1, APE1, CDK3, CDC71, CDK7, CNK, PRL-3,STK2 (NEK4), and HBO1 were identified as modulators of cellproliferation using yeast two hybrid assays known to those of skill inthe art (see, e.g., Fields and Song, Nature, 340(6230):245 (1989).Briefly, two different haploid yeast strains of opposite mating types(e.g., MATa and MATα) are generated. One strain contains a protein fusedto the DNA binding domain (i.e., binds to UASG) of the Saccharomycescerevisiae transcriptional activator factor GAL4. The GAL4 DNA bindingdomain is typically placed upstream of reporter genes. Another straincontains a protein fused to the activation domain of GALA. The strainsare mated and transcription of the reporter gene is assayed. If the twoproteins fused to the GAL4 domains interact to form a protein-proteincomplex, the DNA binding domain and the activation domain willreconstitute to form a functional transcriptional activator and reportergene activity will be detected.

Example 3 Functional Characterization of Genes that Modulate the CellCycle Using Dominant Negative Mutants

[0304] Dominant negative mutants are used to study the effects of PKC-ζ,PLC-β1, FAK, FAK2, CK2, cMET, FEN1, REV1, APE1, CDK3, PIM1, CDC7L1,CDK7, CNK, PRL-3, STK2 or NEK4, NKIAMRE, or HBO1 on proliferation, thecell cycle, cell viability, and chemosensitization.

[0305] The anti-proliferative effects of dominant negative mutants aredetermined by GFP positivity assays. Briefly, Cell Tracker (CT) stainedcells are infected with retroviruses engineered to express wild type andmutant PKC-ζ, PLC-β1, FAK, FAK2, CK2, cMET, FEN1, REV1, APE1, CDK3,PIM1, CDC7L1, CDK7, CNK, PRL-3, STK2 or NEK4, NKIAMRE, or HBO1. The CTintensity of the GFP expressing population will be compared to theintensity of the GFP negative, uninfected population. Cells that stainbrightly with the CT are identified as cell cycle arrested cells. Cellsthat stain dimly with CT are identified as proliferating cells.

[0306] Effects of dominant negative mutants on the cell cycle ismeasured by DAPI staining of transfected cells.

[0307] Effects of dominant negative mutants on cell viability isdetermined by monitoring the percent of GFP positive cells in aninfected population at set intervals following infection.

[0308] Effects of dominant negative mutants on chemosensitization isdetermined by first treating transfected cells with chemotherapeuticagents such as, for example, bleomycin, etoposide, and cisplatin. Aftertreatment with the chemotherapeutic agent, CT assays, DAPI stainingassays, and GFP-positivity assays are conducted to assess the effects ofPKC-ζ, PLC-β1, FAK, FAK2, CK2, cMET, FEN1, REV1, APE1, CDK3, PIM1,CDC7L1, CDK7, CNK, PRL-3, STK2 or NEK4, NKIAMRE, or HBO1 onproliferation, the cell cycle, cell viability, and chemosensitization.

[0309] Dominant negative mutants are used to determine the effects ofPKC-ζ, PLC-β1, FAK, FAK2, CK2, cMET, FEN1, REV1, APE1, CDK3, PIM1,CDC7L1, CDK7, CNK, PRL-3, STK2 or NEK4, NKIAMRE, or HBO1 in differenttumor types such as, for example, lung, colon, cervical, liver, kidney,uterine, or breast. Exemplary tumor cells lines include, A549 cells(lung, p53 wt), H1299 (lung, p53 null), Hela (cervix, p53 deficient),Colo205 (colon, p53 mutant), and HCT116 (colon, p53 wt).

[0310] Dominant negative mutants are also used to determine the effectsof PKC-ζ, PLC-β1, FAK, FAK2, CK2, cMET, FEN1, REV1, APE1, CDK3, PIM1,CDC7L1, CDK7, CNK, PRL-3, STK2 or NEK4, NKIAMRE, or HBO1 in tumor cellsversus normal cells. Exemplary tissue types include mammary epithelialcells, prostate epithelial cells, lung cells, kidney cells, cervicalcells and colon cells.

[0311] Dominant negative mutants were generated for CDC7L1, CNK, STK2,Hbo1, PIM1, APE1, CK2 or CK2α, NKIAMRE, FEN1, and CDK3. The results aredescribed in examples below.

Example 4 Functional Characterization of Genes that Modulate the CellCycle Using siRNA

[0312] Short interfering RNAs (siRNAs) are used to study the effects ofPKC-ζ, PLC-β1, FAK, FAK2, CK2, cMET, FEN1, REV1, APE1, CDK3, PIM1,CDC7L1, CDK7, CNK, PRL-3, STK2 or NEK4, NKIAMRE, or HBO1 onproliferation and chemosensitization.

[0313] Four siRNAs are designed for each gene and transfected into A549cells and Hela cells. mRNA reduction is tested using Taqman. siRNAs thatinduce greater than 70% mRNA reduction are tested for anti-proliferativeeffects. Cy-3 labeled control siRNA, scrambled siRNAs, and thetransfection reagent are used as controls.

[0314] siRNAs which show no independent anti-proliferative effects areanalyzed for their ability to confer chemosensitization. 48 hours posttransfection, cells are treated with chemotherapeutic agents, such as,for example, bleomycin, etoposide, and cisplatin. 48 hourspost-treatment, the IC50 of each chemotherapeutic agent is determinedusing BrdU ELISA and/or Cellomics image analysis which counts coloniesand measures colony size.

[0315] siRNAs were designed for CDC7L1, CNK, Hbo1, PIM1, CK2 or CK2α,and NKIAMRE. The results are discussed in examples below.

Example 5 Functional Characterization of Genes that Modulate the CellCycle Using Antisense Oligonucleotides

[0316] Antisense oligonucleotides are used to study the effects ofPKC-ζ, PLC-β1, FAK, FAK2, CK2, cMET, FEN1, REV1, APE1, CDK3, PIM1,CDC7L1, CDK7, CNK, PRL-3, STK2 or NEK4, NKIAMRE, or HBO1 onproliferation and chemosensitization. Briefly, antisenseoligonucleotides with a mixed phosphothiorate backbone are used totransfect A549 and Hela cells. Oligonucleotide concentrations of 50 nMor 100 nM are used to transfect the cells. Oligonucleotides which inducegreater than 70% mRNA reduction in transfected cells will be tested foranti-proliferative effects. Cell proliferation and viability assays areperformed 48 hours post transfection with a BrdU ELISA and/or Cellomicsimage analysis which counts colonies and measures colony size. Antisenseoligonucleotides which show no independent anti-proliferative effectsare analyzed for their ability to confer chemosensitization. 48 hourspost transfection, cells are treated with chemotherapeutic agents, suchas, for example, bleomycin, etoposide, and cisplatin. 48 hourspost-treatment, the IC50 of each chemotherapeutic agent is determinedusing BrdU ELISA and/or Cellomics image analysis.

[0317] Antisense oligonucleotides are used to determine the effects ofPKC-ζ, PLC-β1, FAK, FAK2, CK2, cMET, FEN1, REV1, APE1, CDK3, PIM1,CDC7L1, CDK7, CNK, PRL-3, STK2 or NEK4, NKIAMRE, or HBO1 in differenttumor types such as, for example, lung, colon, cervical, liver, kidney,uterine, or breast. Exemplary tumor cells lines include, A549 cells(lung, p53 wt), H1299 (lung, p53 null), Hela (cervix, p53 deficient),Colo205 (colon, p53 mutant), and HCT115 (colon, p53 wt).

[0318] Antisense oligonucleotides are also used to determine the effectsof PKC-ζ, PLC-β1, FAK, FAK2, CK2, cMET, FEN1, REV1, APE1, CDK3, PIM1,CDC7L1, CDK7, CNK, PRL-3, STK2 or NEK4, NKIAMRE, or HBO1 in tumor cellsversus normal cells. Exemplary tissue types include mammary epithelialcells, prostate epithelial cells, lung cells, kidney cells, cervicalcells and colon cells.

Example 6 Identification of Genes that Modulate the Cell Cycle UsingProteomics

[0319] Proteomics assays are used to identify proteins that bind toPKC-ζ, PLC-β1, FAK, FAK2, CK2, cMET, FEN1, REV1, APE1, CDK3, PIM1,CDC7L1, CDK7, CNK, PRL-3, STK2 or NEK4, NKIAMRE, or HBO1. Typically, theproteomics assays are performed after a functional screen to identify agene of interest. Briefly, a potential binding partner is mixed with aPKC-ζ, PLC-β1, FAK, FAK2, CK2, cMET, FEN1, REV1, APE1, CDK3, PIM1,CDC7L1, CDK7, CNK, PRL-3, STK2 or NEK4, NKIAMRE, or HBO1 polypeptidebound to an affinity tag (i.e. a labeled monoclonal antibody). Complexesof the potential binding partner bound to the polypeptide are extracted,and analyzed, and the potential binding partner is identified.

Example 7 Assay for PLCβ1 Activity

[0320] PLCβ1 activity can be measured according to the method describedin Nomoto et al., Jpn. J Canc. Res., 89:1257-1266 (1998). Briefly, cellextracts are prepared and an appropriate amount of cell extract issuspended in reaction buffer (50 mM HEPES, pH 7.0, 100 mM NaCl, 1 mMCaCl₂, 0.15 mg/ml bovine serum albumin, and 1 mg/ml sodium deoxycholate)mixed with micelles of a substrate mixture of 1-α-phosphatidyl inositoland 1-α-phosphatidyl [2-³H] inositol or a substrate mixture of1-α-phosphatidyl inositol 4, 5-biphosphate and 1-α-phosphatidyl [2-³H]inositol 4,5-biphosphate at final concentrations of 100 μM and 10⁴ dpm,respectively. After an appropriate incubation, the reaction is stopped,lipids are extracted from the reaction mixture and radioactivity in theaqueous fraction is detected with a liquid scintillation counter.Percent degradation of the labeled substrate is indicative of enzymaticactivity.

Example 8 Assay for FAK2 Activity

[0321] FAK2 protein-tyrosine kinase activity can be measured accordingto the method described in Sasaki et al., J. Bio. Chem., 270(6):21206(1995). Briefly, clarified cell lysates are incubated in 20 μl of kinaseassay buffer with 5 μg/20 μl of poly (Glu,Tyr), 5 μCi of [γ-³²P]ATP, 5μM unlabeled ATP, and 5 M MgCl₂. After an appropriate incubation, thereaction is stopped, and labeled substrate is separated by SDS-PAGE.³²P-phosphorylated poly (Glu,Tyr) is visualized and quantitated bybioimaging analysis.

Example 9 Assay for CK2 Activity

[0322] CK2 activity can be measured according to the method described inMessenger et al., J. Biol. Chem., 277(25):23054 (2002). Briefly, cellextracts are incubated in 1 mM of a synthetic peptide substrate,RRRDDDSDDD in 20 mM Tris-HCl pH 7.5, 60 mM NaCl, 10 mM MgCl₂, 1 mM DTT,and 100 μM γ-32P-ATP. After an appropriate incubation, the reactions arestopped, run on SDS-PAGE, and phosphorylated proteins are detected bybioimaging analysis.

Example 10 Assay for cMET Activity

[0323] cMET activity can be measured according to the method describedin Jeffers et al., Proc. Nat 7. Acad. Sci. USA 94:11445 (1997). Briefly,cell lysates are prepared and immunoprecipitated using anti-Met SP260(Santa Cruz Biotechnology) monoclonal antibody. Immunoprecipitates areassessed or tyrosine kinase activity toward the exogenous substrategastrin using a tyrosine kinase assay kit from Boehringer Mannheim.

Example 11 Assay for FEN1 Activity

[0324] FEN1 activity can be measured according to the method describedin Tom et al., J. Biol. Chem. 275(14):10498 (2000). Briefly, FEN1 ispurified from cell extracts and incubated with appropriate amounts ofoligonucleotide substrates and proliferating cell nuclear antigen inreaction buffer (30 mM HEPES pH 7.6, 5% glycerol, 40 mM KCL, 0.1 mg. mlbovine serum albumin, and 8 mM MgCl₂). After an appropriate incubation,the reactions are stopped, run on SDS-PAGE, and products are detected bybioimaging analysis.

Example 12 Assay for REV1 Activity

[0325] REV1 activity can be measured according to the method describedin Zhang et al., Nuc. Acids Res. 30(7):1630 (2002)). Briefly, REV1 ispurified from cell extracts and incubated in reaction buffer (25 mMKH₂PO₄ pH 7.0, 5 mM MgCl₂, 10% glycerol, and 50 μM of dNTPs (dATP, dCTP,dTTP, and dGTP) and 50 fmol of a DNA substrate containing a 5′- ³²plabeled primer. After an appropriate incubation, the reactions arestopped, run on SDS-PAGE, and products are detected by bioimaginganalysis.

Example 13 Assay for APE1 Activity

[0326] APE1 activity can be measured according to the method describedin Tom et al., J. Biol. Chem., 276(52):48781 (2001). Briefly, APE1 ispurified from cell extracts and incubated with appropriate amounts ofoligonucleotide substrates in reaction buffer (30 mM HEPES pH 7.6, 5%glycerol, 40 mM KCL, 0.01% Nonidet P-40, 1 mg/ml bovine serum albumin, 8mM MgCl₂, and 0.1 mM ATP). After an appropriate incubation, thereactions are stopped, run on SDS-PAGE, and products are detected bybioimaging analysis.

Example 14 Assay for CDC7 L1 Activity

[0327] CDC7L1 activity can be measured according to the method describedin Masai, et al., J. Biol. Chem., 275(37):29042 (2000). BrieflyCDC7L1-ASK complexes are purified, mixed with [γ-32P]ATP (1 μCi) andadded to a reaction mixture containing MCM2-4-6-7-previously incubatedwith cdks and p27. After an appropriate incubation, the reactions arestopped, run on SDS-PAGE, and products are detected by bioimaginganalysis.

Example 15 Assay for CNK Activity

[0328] CNK activity can be measured according to the method described inOuyang et al., J. Biol. Chem. 274:28646 (1997). Briefly, CNK is purifiedand assayed for kinase activity using one or more of the followingsubstrates: casein (15 μg/reaction), p53, GST-Cdc25A (5 μg/reaction),GST-Cdc25B (5 μg/reaction), His6-Cdc25c (5 μg/reaction), GST-Cdc25C (1μg/reaction), or GST-Cdc25C^(S216A) (1 μg/reaction).

Example 16 Assay for STK2 (NEK4) Activity

[0329] STK2 (NEK4) activity can be measured according to the methoddescribed in Hayashi et al., Biochem. Biophys. Res. Comm., 264:449(1999). Briefly, STK2 complexes are immunoprecipitated, resuspended inkinase buffer (50 mM Tris-HCl pH 7.2, 3 mM MnCl₂) containing 10 μCi[γ-32P]ATP and 5 μg of exogenous protein substrates. After anappropriate incubation, the reactions are stopped, the phosphorylatedproteins are separated by SDS-PAGE, and detected by bioimaging analysis.

Example 17 Assay for HBO1 Activity

[0330] HBO1 can be measured according to the method described in Iiuzukaand Stilman, J. Bio. Chem., 274(33):23027 (1999). Briefly, HBO1polypeptides are immunoprecipitated from cell extracts and combined witha mixture recombinant Xenopus histone H3₂.H4₂ tetramers (100 μg/ml),human histone H2A.H2B (100 μg/ml), and pmol of [³H]acetyl coenzyme A(11.2 Ci/mmol) in an appropriate volume of assay buffer (25 mM Tris-HCl,ph 8.5m 1 mM dithiothreitol, 0.5 mM EDTA, 5 mM sodium butyrate, 150 mMNaCl, 10% glycerol). After an appropriate incubation, the reactions arestopped, the phosphorylated proteins are separated by SDS-PAGE, anddetected by Coomassie blue staining.

Example 18 Functional Characterization of CDC7 L1 Using DominantNegative Mutants and siRNA Assays

[0331] CDC7LI was identified as a modulator of cellular proliferation ina yeast two hybrid assay using apoptin and GADD45. Vectors for theexpression of CDC7LI fused to the C-terminus of GFP with a tetOffinducible gene expression system were used to transfect A549 cells andHela cells. Cell proliferation was measured using Cell Tracker assays,i.e., detecting GFP positivity. As shown in FIG. 20, expression ofwild-type GFP-CDC7 LI and mutant GFP-CDC7LI inhibited proliferation ofA549 cells. The amino acid sequence of CDC7L muntants is shown in FIG.26.

[0332] CDC7LI mRNA expression was analyzed in tumor cell lines and inlung carcinomas and colon carcinomas. CDC7LI mRNA was overexpressed intumor cell lines (e.g., DU145, HCT116, SW620, Hela, and PC3) as comparedto primary cell lines. See, e.g., FIG. 27. FIG. 28 demonstrates thatCDC7LI mRNA is expressed at higher levels in some lung carcinomascompared to normal tissue from the same patient. FIG. 29 demonstratesthat CDC7LI mRNA is expressed at higher levels in some colon carcinomascompared to normal tissue from the same patient.

[0333] Two siRNAs induced greater than 50% reduction in mRNA expressionwhen transfected into Hela cells. One of these siRNAs induced greaterthan 70% reduction in mRNA expression. (Data not shown.)

Example 19 Functional Characterization of CNK Using Dominant NegativeMutants and siRNA Assays

[0334] CNK was identified as a modulator of cellular proliferation in ayeast two hybrid assay using DNAPK and F10. Vectors for the expressionof CNK fused to the C-terminus of GFP with a tetOff inducible geneexpression system were used to transfect A549 cells and Hela cells. Cellproliferation was measured using Cell Tracker assays, i.e., detectingGFP positivity. As shown in FIG. 21, expression of wild-type CNK andmutant GFP-CNK inhibited proliferation of A549 cells. None of the siRNAstested induced greater than 50% reduction in mRNA expression.

[0335] CNK mRNA expression was analyzed in tumor cell lines. CNK mRNAwas overexpressed in tumor cell lines (e.g., HCT116, PC3, A549, colo205,and H1299) as compared to primary cell lines. See, e.g., FIG. 30.

[0336] Wild type CNK and the CNK D146A mutant were fused to GST andproduced in E. coli. (Data not shown.) Briefly, BL21(DE3) cells weretransformed with either pDEST15-CNK WT or CNK D146A and grown at 37° C.to an OD600 of 0.6. Cultures were induced with 1 mM IPTG and thentransferred to a 16° C. shaking incubator for overnight incubation.After immobilization on glutathione-sepharose, proteins were eluted with7.5 mM glutathione. The yield was approximately 0.5 mg/L for eachprotein.

[0337] The GST CNK fusions were tested for kinase activity in duplicateassays. See, e.g., FIG. 31. The reaction buffer contained the followingcomponents: Reaction buffer: 10 mM Hepes, 10 μM ATP, 10 μM MnCl₂, 10 μCiγ-³²P ATP, 5 mM MgCl₂, 1 mM DTT, 1 mM Na₃VO₄, 100 ng GST-CNK, 1.2 μg p53or 10 μg MBP. Kinase reactions were incubated for thirty minutes at roomtemperature. The GST-CNK D146A mutant did not exhibit kinase activity.Wild type GST-CNK phosphorylated p53, maltose binding protein (MBP) andalso exhibited autophosphorylation activity.

Example 20 Functional Characterization of STK2 Using Dominant NegativeMutants

[0338] STK2 was identified as a modulator of cellular proliferation in ayeast two hybrid assay using p73. STK2 is expressed as long and shortisoforms (STK2L and STK2S). STK2L appears to be more highly expressedthan STK2S in humans. See, e.g., FIG. 32.

[0339] STK2 mRNA expression was analyzed in tumor cell lines. STK2 mRNAwas overexpressed in tumor cell lines (e.g., HCT116 and PC3) as comparedto primary cell lines. See, e.g., FIG. 33.

[0340] STK2 clones from a GFP C-terminal cDNA fusion library with atetOff inducible gene expression system were used to transfect A549cells and Hela cells. Cell proliferation was measured using Cell Trackerassays, i.e., detecting GFP positivity. As shown in FIG. 22, expressionof wild-type STK2S inhibited proliferation of A549 cells and in Helacells and expression of and mutant STK2S inhibited proliferation of A549cells. Similar results are shown in FIG. 34. FIG. 35 shows thatexpression of GFP-STK2L inhibited proliferation of A549 and HeLa cells.Similar results were obtained for STK2L as shown in FIG. 36. Using IRESvectors, expression of STK2L wild type and mutant proteins inhibitedproliferation in A549 cells. See, e.g., FIG. 37.

Example 21 Functional Characterization of HBO1

[0341] Hbo1 mutants were constructed with the following mutations: Hbo1G484E, Hbo1 L497S, and Hbo1 E508Q. Hbo1 mutants are shown in FIG. 72.Both wild type and mutant Hbo1 proteins were localized to the cellnucleus. (Data not shown.)

[0342] The effect of Hbo1 expression on tumor cell lines was determinedusing cells that had been infected with a retrovirus that expressed HBO1wild type or mutant proteins. The Hbo1 E508Q mutant wasantiproliferative in A549 cells (IRES only) and HeLa cells (GFP fusionand IRES construct) and had no effect in H1299 cells. Expression of thewild type Hbo1 protein and the other mutants had no effect onproliferation in this assay. See, e.g., FIGS. 38-40. Additional assayswere performed using only sorted GFP positive cells as shown in FIG. 41.Proliferation was measured using the CyQuant Cell Proliferation Assay(Molecular Probes) which is based upon the fluorescence enhancement uponbinding of a proprietary dye to cellular DNA. Using sorted cells, theHbo1 E508Q mutant was strongly antiproliferative in A549 cells and HeLacells. See, e.g., FIGS. 42-43.

[0343] An Hbo1 siRNA caused greater than 50% reduction in mRNAexpression when transfected into A549 cells or H1299 cells. The sequenceof the Hbo1 siRNA is as follows: AACTGAGCAAGTGGTTGATTT. The Hbo1 siRNAhad an antiproliferative effect when expressed in A549 or H1299 cells.See, e.g., FIGS. 44-45.

Example 22 Functional Characterization of PIM1

[0344] PIM1 ImRNA expression was analyzed in tumor cell lines andprimary human tumors. PIM1 mRNA was overexpressed in tumor cell lines(e.g., H1299, PC3, DU145, HCC1937, and MDA-MB-231) as compared toprimary cell lines. See, e.g., FIG. 46. PIM1 appeared to be expressed atlower levels in breast carcinomas as compared to normal tissue from thesame patient. See, e.g., FIG. 47. PIM1 also appeared to be expressed atlower levels in lung carcinomas as compared to normal tissue from thesame patient. See, e.g., FIG. 48.

[0345] PIM1 mutants were constructed with the following mutations: Pim1K67A and PIM1 D186N. PIM1 mutants are shown in FIG. 73.

[0346] Vectors for the expression of PIM1 fused to the C-terminus of GFPwith a tetOff inducible gene expression system were used to transfectA549 cells and H1299 cells. Similar experiments were done using an IRESvector. Cell proliferation was measured using Cell Tracker assays, i.e.,detecting GFP positivity. FIG. 49 shows that in A549 cells, expressionof wild type PIM1, but not the mutants, was antiproliferative. FIG. 50shows that in H1299 cells GFP fused wild type PIM1 wasantiproliferative. Using IRES constructs, expression of wild type PIM1and the PIM1 mutants was antiproliferative in H1299 cells.

[0347] A PIM1-specific siRNA caused greater than 50% reduction in mRNAexpression when transfected into A549 cells, HeLa cells, or H1299 cells.The sequence of the PIM1 siRNA is as follows: AAAACTCCGAGTGAACTGGTC. ThePIM1 siRNA had an antiproliferative effect when expressed in A549, HeLacells, or H1299 cells. See, e.g., FIGS. 51-53. In primary HUVEC cellsthe PIM1-specific siRNA caused greater than 50% reduction in mRNAexpression and had an antiproliferative effect. See, e.g., FIG. 54.

[0348] Wild type and mutant PIM1 proteins were expressed in Phoenixcells and assayed for kinase activity using Histone H1 as a substrate.Wild type and mutant PIM1 proteins were fused to GFP and also had a myctag. Wild type and mutant PIM1 proteins were immunoprecipitated using ananti-myc antibody and the immune complexes were assayed for kinaseactivity using 20 μl of kinase buffer+0.5 μL of γ-³²P ATP (3000Ci/mmol). Kinase buffer contained 20 mM Tris, pH 7.5; 50 mM NaCl; 10 mMMgCl₂; 2 mM MnCl₂; 1 mM NaF; and 1 mM Na₃VO₄. Kinase reactions wereincubated at room temperature for one hour and analyzed by SDS-PAGE andautoradiography. Wild type PIM1 exhibited kinase activity, while themutant PIM1 proteins did not. (Data not shown.) Western blot analysiswas used to show the equivalent amounts of wild type and mutant PIM1proteins were assayed. (Data not shown.)

Example 23 Functional Characterization of APE1

[0349] APE1 mutants were constructed with the following mutations: APE1E96A, APE1 D210A, and APE1 C65A.

[0350] Subcellular localization studies demonstrated that APE1 mutantand wild type proteins were localized to the cell nucleus in A549 cells.(Data not shown.)

[0351] Vectors for the expression of APE1 fused to the C-terminus of GFPwith a tetOff inducible gene expression system were used to transfectA549 cells and H1299 cells. APE1 mutants were also expressed. Similarexperiments were done using an IRES vector. Cell proliferation wasmeasured using Cell Tracker assays, i.e., detecting GFP positivity. InA549 cells, expression of wild type and mutant APE1 proteins had noapparent effect on proliferation. See, e.g., FIG. 55. Similar resultswere obtained in H1299 cells. See, e.g., FIG. 56. However, in primaryHMEC cells, expression of both wild type APE1 and the APE1 D210A mutantwas antiproliferative. See, e.g., FIG. 57.

[0352] Expression of the APE1 D210A mutant in A549 cells sensitized thecells to methyl methanesulfonante (MMS) treatment. At 72 hours afterinfection, A549 cells were treated with 3 mM MMS for 60 min. Survivalcurves are shown in FIG. 58.

[0353] Expression of APE1 wildtype and the APE1 C65A mutant wereprotective in A549, HeLa, and H1299 cells treated with bleomycin. See,e.g., FIGS. 59-60. These results are consistent with those published byRobertson et al., Cancer Res. 61:2220-5 (2001), showing thatoverexpression of Ape1 in the tumor line NT2 confers resistance tobleomycin treatment.

Example 24 Functional Characterization of Casein kinase II alpha (CK2αor CK2)

[0354] CK2α mRNA expression was analyzed in tumor cell lines and primaryhuman cell lines and results are shown in FIG. 61. CK2α dominantnegative mutants are shown in FIG. 62. Subcellular localization studiesdemonstrated that CK2α mutant and wild type proteins were localized tothe cell nucleus and concentrated in punctuate areas outside the nucleusin A549 cells. (Data not shown.) Neither CK2α wild type or mutantprotein expression was antiproliferative in A549 or H1299 cells. (Datanot shown.)

[0355] A CK2α-specific siRNA caused greater than 50% reduction in mRNAexpression when transfected into H1299 cells. The sequence of theCK2α-specific siRNA (also know as CK2) is as follows:AACATTGAATTAGATCCACGT. The CK2α siRNA had an antiproliferative effectwhen expressed in H1299 cells. See, e.g., FIG. 63. The same CK2α siRNAreduced mRNA in HeLa cells but did not appear to effect cellproliferation. (Data not shown.)

Example 25 Functional Characterization of NKIAMRE

[0356] NKIAMRE mRNA expression was analyzed in tumor cell lines. NKIAMREmRNA was overexpressed in tumor cell lines (e.g., H1299, PC3, DU145,HCT116, and MDA-MB-231) as compared to primary cell lines. See, e.g.,FIG. 64. Dominant negative mutants of NKIAMRE were generated and areshown in FIG. 65. Subcellular localization studies demonstrated thatNKIAMRE mutant and wild type proteins were localized to the cellcytoplasm in A549 cells. (Data not shown.)

[0357] Vectors for the expression of NKIAMRE fused to the C-terminus ofGFP with a tetOff inducible gene expression system were used totransfect A549 cells and H1299 cells. NKIAMRE mutants were alsoexpressed. Cell proliferation was measured using Cell Tracker assays,i.e., detecting GFP positivity. In A549 cells and H1299 cells,expression of wild type and mutant NKIAMRE proteins had no apparenteffect on proliferation. See, e.g., FIG. 74.

[0358] NKIAMRE-specific siRNA caused greater than 50% reduction in mRNAexpression when transfected into H1299 cells or HeLa cells, but did notappear to affect proliferation in either cell line. Data not shown.

Example 26 Functional Characterization of FEN1

[0359] Dominant negative mutants of FEN1 were generated and are shown inFIG. 66. Vectors for the expression of FEN1 fused to the C-terminus ofGFP with a tetOff inducible gene expression system were used totransfect A549 cells and H1299 cells. GFP fusions were also made usingthe FEN1 dominant negative mutants. Similar experiments were done usingan IRES vector. Cell proliferation was measured using Cell Trackerassays, i.e., detecting GFP positivity. FIG. 67 shows that in A549cells, expression of mutant FEN1, but not the wild type, wasantiproliferative. FIG. 68 shows that in H1299 cells, expression of theFEN1 dominant negative mutants was also antiproliferative.

Example 27 Functional Characterization of CDK3

[0360] Dominant negative mutants of CDK3 were generated and are shown inFIG. 69. Vectors for the expression of CDK3 fused to the C-terminus ofGFP with a tetOff inducible gene expression system were used totransfect A549 cells and H1299 cells. GFP fusions were also made usingthe CDK3 dominant negative mutants. Similar experiments were done usingan IRES vector. Cell proliferation was measured using Cell Trackerassays, i.e., detecting GFP positivity. FIG. 70 shows that in A549cells, expression of either wild type CDK3 or mutant CDK3 proteins hadno apparent antiproliferative effect. FIG. 71 shows that in H1299 cells,expression of either wild type CDK3 or mutant CDK3 proteins had noapparent antiproliferative effect.

[0361] It is understood that the examples and embodiments describedherein are for illustrative purposes only and that various modificationsor changes in light thereof will be suggested to persons skilled in theart and are to be included within the spirit and purview of thisapplication and scope of the appended claims. All publications, patents,and patent applications cited herein are hereby incorporated byreference in their entirety for all purposes.

1 78 1 2164 DNA Homo sapiens protein kinase C, zeta (PKC-zeta), atypicalprotein kinase C isoform 1 atgcccagca ggaccgaccc caagatggaa gggagcggcggccgcgtccg cctcaaggcg 60 cattacgggg gggacatctt catcaccagc gtggacgccgccacgacctt cgaggagctc 120 tgtgaggaag tgagagacat gtgtcgtctg caccagcagcacccgctcac cctcaagtgg 180 gtggacagcg aaggtgaccc ttgcacggtg tcctcccagatggagctgga agaggctttc 240 cgcctggccc gtcagtgcag ggatgaaggc ctcatcattcatgttttccc gagcacccct 300 gagcagcctg gcctgccatg tccgggagaa gacaaatctatctaccgccg gggagccaga 360 agatggagga agctgtaccg tgccaacggc cacctcttccaagccaagcg ctttaacagg 420 agagcgtact gcggtcagtg cagcgagagg atatggggcctcgcgaggca aggctacagg 480 tgcatcaact gcaaactgct ggtccataag cgctgccacggcctcgtccc gctgacctgc 540 aggaagcata tggattctgt catgccttcc caagagcctccagtagacga caagaacgag 600 gacgccgacc ttccttccga ggagacagat ggaattgcttacatttcctc atcccggaag 660 catgacagca ttaaagacga ctcggaggac cttaagccagttatcgatgg gatggatgga 720 atcaaaatct ctcaggggct tgggctgcag gactttgacctaatcagagt catcgggcgc 780 gggagctacg ccaaggttct cctggtgcgg ttgaagaagaatgaccaaat ttacgccatg 840 aaagtggtga agaaagagct ggtgcatgat gacgaggatattgactgggt acagacagag 900 aagcacgtgt ttgagcaggc atccagcaac cccttcctggtcggattaca ctcctgcttc 960 cagacgacaa gtcggttgtt cctggtcatt gagtacgtcaacggcgggga cctgatgttc 1020 cacatgcaga ggcagaggaa gctccctgag gagcacgccaggttctacgc ggccgagatc 1080 tgcatcgccc tcaacttcct gcacgagagg gggatcatctacagggacct gaagctggac 1140 aacgtcctcc tggatgcgga cgggcacatc aagctcacagactacggcat gtgcaaggaa 1200 ggcctgggcc ctggtgacac aacgagcact ttctgcggaaccccgaatta catcgccccc 1260 gaaatcctgc ggggagagga gtacgggttc agcgtggactggtgggcgct gggagtcctc 1320 atgtttgaga tgatggccgg gcgctccccg ttcgacatcatcaccgacaa cccggacatg 1380 aacacagagg actacctttt ccaagtgatc ctggagaagcccatccggat cccccggttc 1440 ctgtccgtca aagcctccca tgttttaaaa ggatttttaaataaggaccc caaagagagg 1500 ctcggctgcc ggccacagac tggattttct gacatcaagtcccacgcgtt cttccgcagc 1560 atagactggg acttgctgga gaagaagcag gcgctccctccattccagcc acagatcaca 1620 gacgactacg gtctggacaa ctttgacaca cagttcaccagcgagcccgt gcagctgacc 1680 ccagacgatg aggatgccat aaagaggatc gaccagtcagagttcgaagg ctttgagtat 1740 atcaacccat tattgctgtc caccgaggag tcggtgtgaggccgcgtgcg tctctgtcgt 1800 ggacacgcgt gattgaccct ttaactgtat ccttaaccaccgcatatgca tgccaggctg 1860 ggcacggctc cgagggcggc cagggacaga cgcttgcgccgagaccgcag agggaagcgt 1920 cagcgggcgc tgctgggagc agaacagtcc ctcacacctggcccggcagg cagcttcgtg 1980 ctggaggaac ttgctgctgt gcctgcgtcg cggcggatccgcggggaccc tgccgagggg 2040 gctgtcatgc ggtttccaag gtgcacattt tccacggaaacagaactcga tgcactgacc 2100 tgctccgcca ggaaagtgag cgtgtagcgt cctgaggaataaaatgttcc gatgaaaaaa 2160 aaaa 2164 2 592 PRT Homo sapiens proteinkinase C, zeta (PKC-zeta), atypical protein kinase C isoform 2 Met ProSer Arg Thr Asp Pro Lys Met Glu Gly Ser Gly Gly Arg Val 1 5 10 15 ArgLeu Lys Ala His Tyr Gly Gly Asp Ile Phe Ile Thr Ser Val Asp 20 25 30 AlaAla Thr Thr Phe Glu Glu Leu Cys Glu Glu Val Arg Asp Met Cys 35 40 45 ArgLeu His Gln Gln His Pro Leu Thr Leu Lys Trp Val Asp Ser Glu 50 55 60 GlyAsp Pro Cys Thr Val Ser Ser Gln Met Glu Leu Glu Glu Ala Phe 65 70 75 80Arg Leu Ala Arg Gln Cys Arg Asp Glu Gly Leu Ile Ile His Val Phe 85 90 95Pro Ser Thr Pro Glu Gln Pro Gly Leu Pro Cys Pro Gly Glu Asp Lys 100 105110 Ser Ile Tyr Arg Arg Gly Ala Arg Arg Trp Arg Lys Leu Tyr Arg Ala 115120 125 Asn Gly His Leu Phe Gln Ala Lys Arg Phe Asn Arg Arg Ala Tyr Cys130 135 140 Gly Gln Cys Ser Glu Arg Ile Trp Gly Leu Ala Arg Gln Gly TyrArg 145 150 155 160 Cys Ile Asn Cys Lys Leu Leu Val His Lys Arg Cys HisGly Leu Val 165 170 175 Pro Leu Thr Cys Arg Lys His Met Asp Ser Val MetPro Ser Gln Glu 180 185 190 Pro Pro Val Asp Asp Lys Asn Glu Asp Ala AspLeu Pro Ser Glu Glu 195 200 205 Thr Asp Gly Ile Ala Tyr Ile Ser Ser SerArg Lys His Asp Ser Ile 210 215 220 Lys Asp Asp Ser Glu Asp Leu Lys ProVal Ile Asp Gly Met Asp Gly 225 230 235 240 Ile Lys Ile Ser Gln Gly LeuGly Leu Gln Asp Phe Asp Leu Ile Arg 245 250 255 Val Ile Gly Arg Gly ThrTyr Ala Lys Val Leu Leu Val Arg Leu Lys 260 265 270 Lys Asn Asp Gln IleTyr Ala Met Lys Val Val Lys Lys Glu Leu Val 275 280 285 His Asp Asp GluAsp Ile Asp Trp Val Gln Thr Glu Lys His Val Phe 290 295 300 Glu Gln AlaSer Ser Asn Pro Phe Leu Val Gly Leu His Ser Cys Phe 305 310 315 320 GlnThr Thr Ser Arg Leu Phe Leu Val Ile Glu Tyr Val Asn Gly Gly 325 330 335Asp Leu Met Phe His Met Gln Arg Gln Arg Lys Leu Pro Glu Glu His 340 345350 Ala Arg Phe Tyr Ala Ala Glu Ile Cys Ile Ala Leu Asn Phe Leu His 355360 365 Glu Arg Gly Ile Ile Tyr Arg Asp Leu Lys Leu Asp Asn Val Leu Leu370 375 380 Asp Ala Asp Gly His Ile Lys Leu Thr Asp Tyr Gly Met Cys LysGlu 385 390 395 400 Gly Leu Gly Pro Gly Asp Thr Thr Ser Thr Phe Cys GlyThr Pro Asn 405 410 415 Tyr Ile Ala Pro Glu Ile Leu Arg Gly Glu Glu TyrGly Phe Ser Val 420 425 430 Asp Trp Trp Ala Leu Gly Val Leu Met Phe GluMet Met Ala Gly Arg 435 440 445 Ser Pro Phe Asp Ile Ile Thr Asp Asn ProAsp Met Asn Thr Glu Asp 450 455 460 Tyr Leu Phe Gln Val Ile Leu Glu LysPro Ile Arg Ile Pro Arg Phe 465 470 475 480 Leu Ser Val Lys Ala Ser HisVal Leu Lys Gly Phe Leu Asn Lys Asp 485 490 495 Pro Lys Glu Arg Leu GlyCys Arg Pro Gln Thr Gly Phe Ser Asp Ile 500 505 510 Lys Ser His Ala PhePhe Arg Ser Ile Asp Trp Asp Leu Leu Glu Lys 515 520 525 Lys Gln Ala LeuPro Pro Phe Gln Pro Gln Ile Thr Asp Asp Tyr Gly 530 535 540 Leu Asp AsnPhe Asp Thr Gln Phe Thr Ser Glu Pro Val Gln Leu Thr 545 550 555 560 ProAsp Asp Glu Asp Ala Ile Lys Arg Ile Asp Gln Ser Glu Phe Glu 565 570 575Gly Phe Glu Tyr Ile Asn Pro Leu Leu Leu Ser Thr Glu Glu Ser Val 580 585590 3 3663 DNA Homo sapiens phosphoinositide-specific phospholipase Cbeta 1, isoform a (PLC-beta1), transcript variant 1 3 cagatggccggggctcaacc cggagtgcac gccttgcaac tcaagcccgt gtgcgtgtcc 60 gacagcctcaagaagggcac caaattcgtc aagtgggatg atgattcaac tattgttact 120 ccaattattttgaggactga ccctcaggga tttttctttt actggacaga tcaaaacaag 180 gagacagagctactggatct cagccttgtc aaagatgcca gatgtgggag acacgccaaa 240 gctcccaaggaccccaaatt acgtgaactt ttggatgtgg ggaacatcgg gcgcctggag 300 cagcgcatgatcacagtggt gtatgggcct gacctcgtga acatctccca tttgaatctc 360 gtggctttccaagaagaagt ggccaaggaa tggacaaatg aggttttcag tttggcaaca 420 aacctgctggcccaaaacat gtccagggat gcatttctgg aaaaagccta tactaaactt 480 aagctgcaagtcactccaga agggcgtatt cctctcaaaa acatatatcg cttgttttca 540 gcagatcggaagcgagttga aactgcttta gaggcttgta gtcttccatc ttcaaggaat 600 gattcaatacctcaagaaga tttcactcca gaagtgtaca gagttttcct caacaacctt 660 tgccctcgacctgaaattga taacatcttt tcagaatttg gtgcaaaaag caaaccatat 720 cttaccgttgatcagatgat ggattttatc aaccttaagc agcgagatcc tcggcttaat 780 gaaatactttatccacctct aaaacaagag caagtccaag tattgattga gaagtatgaa 840 cccaacaacagcctcgccag aaaaggacaa atatcagtgg atgggttcat gcgctatctg 900 agtggagaagaaaacggagt cgtttcacct gagaaactgg atttgaatga agacatgtct 960 cagcccctttctcactattt cattaattcc tcgcacaaca cctacctcac agctggccaa 1020 ctggctggaaactcctctgt tgagatgtat cgccaagtgc tcctgtctgg ttgtcgctgt 1080 gtggagctggactgctggaa gggacggact gcagaagagg aacctgtcat cacccatggc 1140 ttcaccatgacaactgaaat atctttcaag gaagtgatag aagcaattgc ggagtgtgca 1200 tttaagacttcaccttttcc aattctcctt tcgtttgaga accatgtgga ttccccaaag 1260 cagcaagccaagatggcgga gtactgccga ctgatctttg gggatgccct tctcatggag 1320 cccctggaaaaatatccact ggaatctgga gttcctcttc caagccctat ggatttaatg 1380 tataaaattttggtgaaaaa taagaagaaa tcacacaagt catcagaagg aagcggcaaa 1440 aagaagctctcagaacaagc ctccaacacc tacagtgact cctccagcat gttcgagccc 1500 tcatccccaggagccggaga agctgatacg gaaagtgacg acgacgatga tgatgatgac 1560 tgtaaaaaatcttcaatgga tgaggggact gctggaagtg aggctatggc cacagaagaa 1620 atgtctaatctggtgaacta tattcagcca gtcaagtttg agtcatttga aatttcaaaa 1680 aaaagaaataaaagttttga aatgtcttcc ttcgtggaaa ccaaaggact tgaacaactc 1740 accaagtctccagtggaatt tgtagaatat aacaaaatgc agcttagcag gatatatcca 1800 aaaggaacacgtgtggattc atccaactat atgcctcagc tcttctggaa tgcaggttgt 1860 cagatggtggcacttaattt ccagacaatg gacctggcta tgcaaataaa tatggggatg 1920 tatgaatacaacgggaagag tggctacaga ttgaagccag agttcatgag gaggcctgac 1980 aagcattttgatccatttac tgaaggcatc gtagatggga tagtggcaaa cactttgtct 2040 gttaagattatttcaggtca gtttctttct gataagaaag ttgggactta cgtggaagta 2100 gatatgtttggtttgcctgt ggatacaagg aggaaggcat ttaagaccaa aacatcccaa 2160 ggaaatgctgtgaatcctgt ctgggaagaa gaacctattg tgttcaaaaa ggtggttctt 2220 cctactctggcctgtttgag aatagcagtt tatgaagaag gaggtaaatt cattggccac 2280 cgtatcttgccagtgcaagc cattcggcca ggctatcact atatctgtct aaggaatgaa 2340 aggaaccagcctctgacgct gcctgctgtc tttgtctaca tagaagtgaa agactatgtg 2400 ccagacacatatgcagatgt catcgaagct ttatcaaacc caatccgata tgtgaacctg 2460 atggaacagagagctaagca attggctgct ttgacactgg aagatgaaga agaagtaaag 2520 aaagaggctgatcctggaga aacaccatca gaggctccaa gtgaagcgag aacgactcca 2580 gcagaaaatggggtgaatca cactacaacc ctgacaccca agccaccctc ccaggctctc 2640 cacagccagccagctccagg ttctgtaaag gcacctgcca aaacagaaga tcttattcag 2700 agtgtcttaacagaagtgga agcacagacc atcgaagaac taaagcaaca gaaatcgttt 2760 gtgaaacttcaaaagaaaca ctacaaagaa atgaaagacc tggttaagag acaccacaag 2820 aaaaccactgaccttatcaa agaacacact accaagtata atgaaattca gaatgactac 2880 ttgagaaggagagccgcttt ggaaaagtcc gccaaaaagg acagtaagaa aaaatcggaa 2940 cccagcagccctgatcatgg ttcatcaacg attgagcaag acctcgctgc tctggatgct 3000 gaaatgacccaaaagttaat agacttgaag gacaaacaac agcagcagct gcttaatctt 3060 cggcaagaacagtattatag tgaaaaatac cagaagcgag aacatattaa actgcttatt 3120 caaaagttgacggatgtcgc agaagagtgt cagaacaatc agttaaagaa gctcaaagaa 3180 atctgtgagaaagaaaagaa agaattaaag aagaaaatgg ataaaaagag gcaggagaag 3240 ataacagaagctaaatccaa agacaaaagt cagatggaag aggagaagac agagatgatc 3300 cggtcatatatccaggaagt ggtgcagtat atcaagaggc tagaagaagc gcaaagtaaa 3360 cggcaagaaaaactcgtaga gaaacacaag gaaatacgtc agcagatcct ggatgaaaag 3420 cccaagctgcaggtggagct ggagcaagaa taccaagaca aattcaaaag actgcccctc 3480 gagattttggaattcgtgca ggaagccatg aaaggaaaga tcagtgaaga cagcaatcac 3540 ggttctgcccctctctccct gtcctcagac cctggaaaag tgaaccacaa gactccctcc 3600 agtgaggagctgggaggaga catcccagga aaagaatttg atactcctct gtgaatgctc 3660 ctg 3663 41216 PRT Homo sapiens phosphoinositide-specific phospholipase C beta 1,isoform a (PLC-beta1), transcript variant 1 4 Met Ala Gly Ala Gln ProGly Val His Ala Leu Gln Leu Lys Pro Val 1 5 10 15 Cys Val Ser Asp SerLeu Lys Lys Gly Thr Lys Phe Val Lys Trp Asp 20 25 30 Asp Asp Ser Thr IleVal Thr Pro Ile Ile Leu Arg Thr Asp Pro Gln 35 40 45 Gly Phe Phe Phe TyrTrp Thr Asp Gln Asn Lys Glu Thr Glu Leu Leu 50 55 60 Asp Leu Ser Leu ValLys Asp Ala Arg Cys Gly Arg His Ala Lys Ala 65 70 75 80 Pro Lys Asp ProLys Leu Arg Glu Leu Leu Asp Val Gly Asn Ile Gly 85 90 95 Arg Leu Glu GlnArg Met Ile Thr Val Val Tyr Gly Pro Asp Leu Val 100 105 110 Asn Ile SerHis Leu Asn Leu Val Ala Phe Gln Glu Glu Val Ala Lys 115 120 125 Glu TrpThr Asn Glu Val Phe Ser Leu Ala Thr Asn Leu Leu Ala Gln 130 135 140 AsnMet Ser Arg Asp Ala Phe Leu Glu Lys Ala Tyr Thr Lys Leu Lys 145 150 155160 Leu Gln Val Thr Pro Glu Gly Arg Ile Pro Leu Lys Asn Ile Tyr Arg 165170 175 Leu Phe Ser Ala Asp Arg Lys Arg Val Glu Thr Ala Leu Glu Ala Cys180 185 190 Ser Leu Pro Ser Ser Arg Asn Asp Ser Ile Pro Gln Glu Asp PheThr 195 200 205 Pro Glu Val Tyr Arg Val Phe Leu Asn Asn Leu Cys Pro ArgPro Glu 210 215 220 Ile Asp Asn Ile Phe Ser Glu Phe Gly Ala Lys Ser LysPro Tyr Leu 225 230 235 240 Thr Val Asp Gln Met Met Asp Phe Ile Asn LeuLys Gln Arg Asp Pro 245 250 255 Arg Leu Asn Glu Ile Leu Tyr Pro Pro LeuLys Gln Glu Gln Val Gln 260 265 270 Val Leu Ile Glu Lys Tyr Glu Pro AsnAsn Ser Leu Ala Arg Lys Gly 275 280 285 Gln Ile Ser Val Asp Gly Phe MetArg Tyr Leu Ser Gly Glu Glu Asn 290 295 300 Gly Val Val Ser Pro Glu LysLeu Asp Leu Asn Glu Asp Met Ser Gln 305 310 315 320 Pro Leu Ser His TyrPhe Ile Asn Ser Ser His Asn Thr Tyr Leu Thr 325 330 335 Ala Gly Gln LeuAla Gly Asn Ser Ser Val Glu Met Tyr Arg Gln Val 340 345 350 Leu Leu SerGly Cys Arg Cys Val Glu Leu Asp Cys Trp Lys Gly Arg 355 360 365 Thr AlaGlu Glu Glu Pro Val Ile Thr His Gly Phe Thr Met Thr Thr 370 375 380 GluIle Ser Phe Lys Glu Val Ile Glu Ala Ile Ala Glu Cys Ala Phe 385 390 395400 Lys Thr Ser Pro Phe Pro Ile Leu Leu Ser Phe Glu Asn His Val Asp 405410 415 Ser Pro Lys Gln Gln Ala Lys Met Ala Glu Tyr Cys Arg Leu Ile Phe420 425 430 Gly Asp Ala Leu Leu Met Glu Pro Leu Glu Lys Tyr Pro Leu GluSer 435 440 445 Gly Val Pro Leu Pro Ser Pro Met Asp Leu Met Tyr Lys IleLeu Val 450 455 460 Lys Asn Lys Lys Lys Ser His Lys Ser Ser Glu Gly SerGly Lys Lys 465 470 475 480 Lys Leu Ser Glu Gln Ala Ser Asn Thr Tyr SerAsp Ser Ser Ser Met 485 490 495 Phe Glu Pro Ser Ser Pro Gly Ala Gly GluAla Asp Thr Glu Ser Asp 500 505 510 Asp Asp Asp Asp Asp Asp Asp Cys LysLys Ser Ser Met Asp Glu Gly 515 520 525 Thr Ala Gly Ser Glu Ala Met AlaThr Glu Glu Met Ser Asn Leu Val 530 535 540 Asn Tyr Ile Gln Pro Val LysPhe Glu Ser Phe Glu Ile Ser Lys Lys 545 550 555 560 Arg Asn Lys Ser PheGlu Met Ser Ser Phe Val Glu Thr Lys Gly Leu 565 570 575 Glu Gln Leu ThrLys Ser Pro Val Glu Phe Val Glu Tyr Asn Lys Met 580 585 590 Gln Leu SerArg Ile Tyr Pro Lys Gly Thr Arg Val Asp Ser Ser Asn 595 600 605 Tyr MetPro Gln Leu Phe Trp Asn Ala Gly Cys Gln Met Val Ala Leu 610 615 620 AsnPhe Gln Thr Met Asp Leu Ala Met Gln Ile Asn Met Gly Met Tyr 625 630 635640 Glu Tyr Asn Gly Lys Ser Gly Tyr Arg Leu Lys Pro Glu Phe Met Arg 645650 655 Arg Pro Asp Lys His Phe Asp Pro Phe Thr Glu Gly Ile Val Asp Gly660 665 670 Ile Val Ala Asn Thr Leu Ser Val Lys Ile Ile Ser Gly Gln PheLeu 675 680 685 Ser Asp Lys Lys Val Gly Thr Tyr Val Glu Val Asp Met PheGly Leu 690 695 700 Pro Val Asp Thr Arg Arg Lys Ala Phe Lys Thr Lys ThrSer Gln Gly 705 710 715 720 Asn Ala Val Asn Pro Val Trp Glu Glu Glu ProIle Val Phe Lys Lys 725 730 735 Val Val Leu Pro Thr Leu Ala Cys Leu ArgIle Ala Val Tyr Glu Glu 740 745 750 Gly Gly Lys Phe Ile Gly His Arg IleLeu Pro Val Gln Ala Ile Arg 755 760 765 Pro Gly Tyr His Tyr Ile Cys LeuArg Asn Glu Arg Asn Gln Pro Leu 770 775 780 Thr Leu Pro Ala Val Phe ValTyr Ile Glu Val Lys Asp Tyr Val Pro 785 790 795 800 Asp Thr Tyr Ala AspVal Ile Glu Ala Leu Ser Asn Pro Ile Arg Tyr 805 810 815 Val Asn Leu MetGlu Gln Arg Ala Lys Gln Leu Ala Ala Leu Thr Leu 820 825 830 Glu Asp GluGlu Glu Val Lys Lys Glu Ala Asp Pro Gly Glu Thr Pro 835 840 845 Ser GluAla Pro Ser Glu Ala Arg Thr Thr Pro Ala Glu Asn Gly Val 850 855 860 AsnHis Thr Thr Thr Leu Thr Pro Lys Pro Pro Ser Gln Ala Leu His 865 870 875880 Ser Gln Pro Ala Pro Gly Ser Val Lys Ala Pro Ala Lys Thr Glu Asp 885890 895 Leu Ile Gln Ser Val Leu Thr Glu Val Glu Ala Gln Thr Ile Glu Glu900 905 910 Leu Lys Gln Gln Lys Ser Phe Val Lys Leu Gln Lys Lys His TyrLys 915 920 925 Glu Met Lys Asp Leu Val Lys Arg His His Lys Lys Thr ThrAsp Leu 930 935 940 Ile Lys Glu His Thr Thr Lys Tyr Asn Glu Ile Gln AsnAsp Tyr Leu 945 950 955 960 Arg Arg Arg Ala Ala Leu Glu Lys Ser Ala LysLys Asp Ser Lys Lys 965 970 975 Lys Ser Glu Pro Ser Ser Pro Asp His GlySer Ser Thr Ile Glu Gln 980 985 990 Asp Leu Ala Ala Leu Asp Ala Glu MetThr Gln Lys Leu Ile Asp Leu 995 1000 1005 Lys Asp Lys Gln Gln Gln GlnLeu Leu Asn Leu Arg Gln Glu Gln Tyr 1010 1015 1020 Tyr Ser Glu Lys TyrGln Lys Arg Glu His Ile Lys Leu Leu Ile Gln 1025 1030 1035 1040 Lys LeuThr Asp Val Ala Glu Glu Cys Gln Asn Asn Gln Leu Lys Lys 1045 1050 1055Leu Lys Glu Ile Cys Glu Lys Glu Lys Lys Glu Leu Lys Lys Lys Met 10601065 1070 Asp Lys Lys Arg Gln Glu Lys Ile Thr Glu Ala Lys Ser Lys AspLys 1075 1080 1085 Ser Gln Met Glu Glu Glu Lys Thr Glu Met Ile Arg SerTyr Ile Gln 1090 1095 1100 Glu Val Val Gln Tyr Ile Lys Arg Leu Glu GluAla Gln Ser Lys Arg 1105 1110 1115 1120 Gln Glu Lys Leu Val Glu Lys HisLys Glu Ile Arg Gln Gln Ile Leu 1125 1130 1135 Asp Glu Lys Pro Lys LeuGln Val Glu Leu Glu Gln Glu Tyr Gln Asp 1140 1145 1150 Lys Phe Lys ArgLeu Pro Leu Glu Ile Leu Glu Phe Val Gln Glu Ala 1155 1160 1165 Met LysGly Lys Ile Ser Glu Asp Ser Asn His Gly Ser Ala Pro Leu 1170 1175 1180Ser Leu Ser Ser Asp Pro Gly Lys Val Asn His Lys Thr Pro Ser Ser 11851190 1195 1200 Glu Glu Leu Gly Gly Asp Ile Pro Gly Lys Glu Phe Asp ThrPro Leu 1205 1210 1215 5 3052 DNA Homo sapiens cytoplasmic tyrosinekinase focal adhesion kinase (FAK) 5 ccggtgtgaa ggccatgagt gattactgggttgttggaaa gaagtctaac tatgaagtat 60 tagaaaaaga tgttggttta aagcgattttttcctaagag tttactggat tctgtcaagg 120 ccaaaacact aagaaaactg atccaacaaacatttagaca atttgccaac cttaatagag 180 aagaaagtat tctgaaattc tttgagatcctgtctccagt ctacagattt gataaggaat 240 gcttcaagtg tgctcttggt tcaagctggattatttcagt ggaactggca atcggcccag 300 aagaaggaat cagttaccta acggacaagggctgcaatcc cacacatctt gctgacttca 360 ctcaagtgca aaccattcag tattcaaacagtgaagacaa ggacagaaaa ggaatgctac 420 aactaaaaat agcaggtgca cccgagcctctgacagtgac ggcaccatcc ctaaccattg 480 cggagaatat ggctgaccta atagatgggtactgccggct ggtgaatgga acctcgcagt 540 catttatcat cagacctcag aaagaaggtgaacgggcttt gccatcaata ccaaagttgg 600 ccaacagcga aaagcaaggc atgcggacacacgccgtctc tgtgtcagaa acagatgatt 660 atgctgagat tatagatgaa gaagatacttacaccatgcc ctcaaccagg gattatgaga 720 ttcaaagaga aagaatagaa cttggacgatgtattggaga aggccaattt ggagatgtac 780 atcaaggcat ttatatgagt ccagagaatccagctttggc ggttgcaatt aaaacatgta 840 aaaactgtac ttcggacagc gtgagagagaaatttcttca agaagcctgc cattacacat 900 ctttgcactg gaattggtgc agatatataagtgatcctaa tgttgatgcc tgcccagacc 960 ccaggaatgc agagttaaca atgcgtcagtttgaccatcc tcatattgtg aagctgattg 1020 gagtcatcac agagaatcct gtctggataatcatggagct gtgcacactt ggagagctga 1080 ggtcattttt gcaagtaagg aaatacagtttggatctagc atctttgatc ctgtatgcct 1140 atcagcttag tacagctctt gcatatctagagagcaaaag atttgtacac agggacattg 1200 ctgctcggaa tgttctggtg tcctcaaatgattgtgtaaa attaggagac tttggattat 1260 cccgatatat ggaagatagt acttactacaaagcttccaa aggaaaattg cctattaaat 1320 ggatggctcc agagtcaatc aattttcgacgttttacctc agctagtgac gtatggatgt 1380 ttggtgtgtg tatgtgggag atactgatgcatggtgtgaa gccttttcaa ggagtgaaga 1440 acaatgatgt aatcggtcga attgaaaatggggaaagatt accaatgcct ccaaattgtc 1500 ctcctaccct ctacagcctt atgacgaaatgctgggccta tgaccccagc aggcggccca 1560 ggtttactga acttaaagct cagctcagcacaatcctgga ggaagagaag gctcagcaag 1620 aagagcgcat gaggatggag tccagaagacaggccacagt gtcctgggac tccggagggt 1680 ctgatgaagc accgcccaag cccagcagaccgggttatcc cagtccgagg tccagcgaag 1740 gattttatcc cagcccacag cacatggtacaaaccaatca ttaccaggtt tctggctacc 1800 ctggttcaca tggaatcaca gccatggctggcagcatcta tccaggtcag gcatctcttt 1860 tggaccaaac agattcatgg aatcatagatctcaggagat agcaatgtgg cagcccaatg 1920 tggaggactc tacagtattg gacctgcgagggattgggca agtgttgcca acccatctga 1980 tggaagagcg tctaatccga cagcaacaggaaatggaaga agatcagcgc tggctggaaa 2040 aagaggaaag atttctgatt ggaaaccaacatatatatca gcctgtgggt aaaccagatc 2100 ctgcagctcc accaaagaaa ccgcctcgccctggagctcc cggtcatctg ggaagccttg 2160 ccagcctcag cagccctgct gacagctacaacgagggtgt caagcttcag ccccaggaaa 2220 tcagcccccc tcctactgcc aacctggaccggtcgaatga taaggtgtac gagaatgtga 2280 cgggcctggt gaaagctgtc atcgagatgtccagtaaaat ccagccagcc ccaccagagg 2340 agtatgtccc tatggtgaag gaagtcggcttggccctgag gacattattg gccactgtgg 2400 atgagaccat tcccctccta ccagccagcacccaccgaga gattgagatg gcacagaagc 2460 tattgaactc tgacctgggt gagctcatcaacaagatgaa actggcccag cagtatgtca 2520 tgaccagcct ccagcaagag tacaaaaagcaaatgctgac tgccgctcac gccctggctg 2580 tggatgccaa aaacttactc gatgtcattgaccaagcaag actgaaaatg cttgggcaga 2640 cgagaccaca ctgagcctcc cctaggagcacgtcttgcta ccctcttttg aagatgttct 2700 ctagccttcc accagcagcg aggaattaaccctgtgtcct cagtcgccag cactcacagc 2760 tccaactttt ttgaatgacc atctggttgaaaaatctttc tcatataagt ttaaccacac 2820 tttgatttgg gttcattttt tgttttgtttttttcaatca tgatattcag aaaaatccag 2880 gatccaaaat gtggcgtttt tctaagaatgaaaattatat gtaagctttt aagcatcatg 2940 aagaacaatt tatgttcaca ttaagatacgttctaaaggg ggatggccaa ggggtgacat 3000 cttaattcct aaactacctt agctgcatagtggaagagga gagccggaat tc 3052 6 879 PRT Homo sapiens cytoplasmictyrosine kinase focal adhesion kinase (FAK) 6 Met Ser Asp Tyr Trp ValVal Gly Lys Lys Ser Asn Tyr Glu Val Leu 1 5 10 15 Glu Lys Asp Val GlyLeu Lys Arg Phe Phe Pro Lys Ser Leu Leu Asp 20 25 30 Ser Val Lys Ala LysThr Leu Arg Lys Leu Ile Gln Gln Thr Phe Arg 35 40 45 Gln Phe Ala Asn LeuAsn Arg Glu Glu Ser Ile Leu Lys Phe Phe Glu 50 55 60 Ile Leu Ser Pro ValTyr Arg Phe Asp Lys Glu Cys Phe Lys Cys Ala 65 70 75 80 Leu Gly Ser SerTrp Ile Ile Ser Val Glu Leu Ala Ile Gly Pro Glu 85 90 95 Glu Gly Ile SerTyr Leu Thr Asp Lys Gly Cys Asn Pro Thr His Leu 100 105 110 Ala Asp PheThr Gln Val Gln Thr Ile Gln Tyr Ser Asn Ser Glu Asp 115 120 125 Lys AspArg Lys Gly Met Leu Gln Leu Lys Ile Ala Gly Ala Pro Glu 130 135 140 ProLeu Thr Val Thr Ala Pro Ser Leu Thr Ile Ala Glu Asn Met Ala 145 150 155160 Asp Leu Ile Asp Gly Tyr Cys Arg Leu Val Asn Gly Thr Ser Gln Ser 165170 175 Phe Ile Ile Arg Pro Gln Lys Glu Gly Glu Arg Ala Leu Pro Ser Ile180 185 190 Pro Lys Leu Ala Asn Ser Glu Lys Gln Gly Met Arg Thr His AlaVal 195 200 205 Ser Val Ser Glu Thr Asp Asp Tyr Ala Glu Ile Ile Asp GluGlu Asp 210 215 220 Thr Tyr Thr Met Pro Ser Thr Arg Asp Tyr Glu Ile GlnArg Glu Arg 225 230 235 240 Ile Glu Leu Gly Arg Cys Ile Gly Glu Gly GlnPhe Gly Asp Val His 245 250 255 Gln Gly Ile Tyr Met Ser Pro Glu Asn ProAla Leu Ala Val Ala Ile 260 265 270 Lys Thr Cys Lys Asn Cys Thr Ser AspSer Val Arg Glu Lys Phe Leu 275 280 285 Gln Glu Ala Cys His Tyr Thr SerLeu His Trp Asn Trp Cys Arg Tyr 290 295 300 Ile Ser Asp Pro Asn Val AspAla Cys Pro Asp Pro Arg Asn Ala Glu 305 310 315 320 Leu Thr Met Arg GlnPhe Asp His Pro His Ile Val Lys Leu Ile Gly 325 330 335 Val Ile Thr GluAsn Pro Val Trp Ile Ile Met Glu Leu Cys Thr Leu 340 345 350 Gly Glu LeuArg Ser Phe Leu Gln Val Arg Lys Tyr Ser Leu Asp Leu 355 360 365 Ala SerLeu Ile Leu Tyr Ala Tyr Gln Leu Ser Thr Ala Leu Ala Tyr 370 375 380 LeuGlu Ser Lys Arg Phe Val His Arg Asp Ile Ala Ala Arg Asn Val 385 390 395400 Leu Val Ser Ser Asn Asp Cys Val Lys Leu Gly Asp Phe Gly Leu Ser 405410 415 Arg Tyr Met Glu Asp Ser Thr Tyr Tyr Lys Ala Ser Lys Gly Lys Leu420 425 430 Pro Ile Lys Trp Met Ala Pro Glu Ser Ile Asn Phe Arg Arg PheThr 435 440 445 Ser Ala Ser Asp Val Trp Met Phe Gly Val Cys Met Trp GluIle Leu 450 455 460 Met His Gly Val Lys Pro Phe Gln Gly Val Lys Asn AsnAsp Val Ile 465 470 475 480 Gly Arg Ile Glu Asn Gly Glu Arg Leu Pro MetPro Pro Asn Cys Pro 485 490 495 Pro Thr Leu Tyr Ser Leu Met Thr Lys CysTrp Ala Tyr Asp Pro Ser 500 505 510 Arg Arg Pro Arg Phe Thr Glu Leu LysAla Gln Leu Ser Thr Ile Leu 515 520 525 Glu Glu Glu Lys Ala Gln Gln GluGlu Arg Met Arg Met Glu Ser Arg 530 535 540 Arg Gln Ala Thr Val Ser TrpAsp Ser Gly Gly Ser Asp Glu Ala Pro 545 550 555 560 Pro Lys Pro Ser ArgPro Gly Tyr Pro Ser Pro Arg Ser Ser Glu Gly 565 570 575 Phe Tyr Pro SerPro Gln His Met Val Gln Thr Asn His Tyr Gln Val 580 585 590 Ser Gly TyrPro Gly Ser His Gly Ile Thr Ala Met Ala Gly Ser Ile 595 600 605 Tyr ProGly Gln Ala Ser Leu Leu Asp Gln Thr Asp Ser Trp Asn His 610 615 620 ArgSer Gln Glu Ile Ala Met Trp Gln Pro Asn Val Glu Asp Ser Thr 625 630 635640 Val Leu Asp Leu Arg Gly Ile Gly Gln Val Leu Pro Thr His Leu Met 645650 655 Glu Glu Arg Leu Ile Arg Gln Gln Gln Glu Met Glu Glu Asp Gln Arg660 665 670 Trp Leu Glu Lys Glu Glu Arg Phe Leu Ile Gly Asn Gln His IleTyr 675 680 685 Gln Pro Val Gly Lys Pro Asp Pro Ala Ala Pro Pro Lys LysPro Pro 690 695 700 Arg Pro Gly Ala Pro Gly His Leu Gly Ser Leu Ala SerLeu Ser Ser 705 710 715 720 Pro Ala Asp Ser Tyr Asn Glu Gly Val Lys LeuGln Pro Gln Glu Ile 725 730 735 Ser Pro Pro Pro Thr Ala Asn Leu Asp ArgSer Asn Asp Lys Val Tyr 740 745 750 Glu Asn Val Thr Gly Leu Val Lys AlaVal Ile Glu Met Ser Ser Lys 755 760 765 Ile Gln Pro Ala Pro Pro Glu GluTyr Val Pro Met Val Lys Glu Val 770 775 780 Gly Leu Ala Leu Arg Thr LeuLeu Ala Thr Val Asp Glu Thr Ile Pro 785 790 795 800 Leu Leu Pro Ala SerThr His Arg Glu Ile Glu Met Ala Gln Lys Leu 805 810 815 Leu Asn Ser AspLeu Gly Glu Leu Ile Asn Lys Met Lys Leu Ala Gln 820 825 830 Gln Tyr ValMet Thr Ser Leu Gln Gln Glu Tyr Lys Lys Gln Met Leu 835 840 845 Thr AlaAla His Ala Leu Ala Val Asp Ala Lys Asn Leu Leu Asp Val 850 855 860 IleAsp Gln Ala Arg Leu Lys Met Leu Gly Gln Thr Arg Pro His 865 870 875 74089 DNA Homo sapiens calcium dependent tyrosine kinase focal adhesionkinase 2 (FAK2) 7 gaattccgtc agccctttta ctcagccaca gcctccggag ccgttgcacacctacctgcc 60 cggccgactt acctgtactt gccgccgtcc cggctcacct ggcggtgcccgaggagtagt 120 cgctggagtc cgcgcctccc tgggactgca atgtgccgat cttagctgctgcctgagagg 180 atgtctgggg tgtccgagcc cctgagtcga gtaaagttgg gcacgttacgccggcctgaa 240 ggccctgcag agcccatggt ggtggtacca gtagatgtgg aaaaggaggacgtgcgtatc 300 ctcaaggtct gcttctatag caacagcttc aatcctggga aaaacttcaaactggtcaaa 360 tgcactgtcc agacggagat ccgggagatc atcacctcca tcctgctgagcgggcggatc 420 gggcccaaca tccggttggc tgagtgctat gggctgaggc tgaagcacatgaagtccgat 480 gagatccact ggctgcaccc acagatgacg gtgggtgagg tgcaggacaagtatgagtgt 540 ctgcacgtgg aagccgagtg gaggtatgac cttcaaatcc gctacttgccagaagacttc 600 atggagagcc tgaaggagga caggaccacg ctgctctatt tttaccaacagctccggaac 660 gactacatgc agcgctacgc cagcaaggtc agcgagggca tggccctgcagctgggctgc 720 ctggagctca ggcggttctt caaggatatg ccccacaatg cacttgacaagaagtccaac 780 ttcgagctcc tagaaaagga agtggggctg gacttgtttt tcccaaagcagatgcaggag 840 aacttaaagc ccaaacagtt ccggaagatg atccagcaga ccttccagcagtacgcctcg 900 ctcagggagg aggagtgcgt catgaagttc ttcaacactc tcgccccgttcgccaacatc 960 gaccaggaga cctaccgctg tgaactcatt caaggatgga acattactgtggacctggtc 1020 attggcccta aagggatccg ccagctgact agtcaggacg caaagcccacctgcctggcc 1080 gagttcaagc agatcaggtc catcaggtgc ctcccgctgg aggagggccaggcagtactt 1140 cagctgggca ttgaaggtgc cccccaggcc ttgtccatca aaacctcatccctagcagag 1200 gctgagaaca tggctgacct catagacggc tactgccggc tgcagggtgagcaccaaggc 1260 tctctcatca tccatcctag gaaagatggt gagaagcgga acagcctgccccagatcccc 1320 atgctaaacc tggaggcccg gcggtcccac ctctcagaga gctgcagcatagagtcagac 1380 atctacgcag agattcccga cgaaaccctg cgaaggcccg gaggtccacagtatggcatt 1440 gcccgtgaag atgtggtcct gaatcgtatt cttggggaag gcttttttggggaggtctat 1500 gaaggtgtct acacaaatca taaaggggag aaaatcaatg tagctgtcaagacctgcaag 1560 aaagactgca ctctggacaa caaggagaag ttcatgagcg aggcagtgatcatgaagaac 1620 ctcgaccacc cgcacatcgt gaagctgatc ggcatcattg aagaggagcccacctggatc 1680 atcatggaat tgtatcccta tggggagctg ggccactacc tggagcggaacaagaactcc 1740 ctgaaggtgc tcaccctcgt gctgtactca ctgcagatat gcaaagccatggcctacctg 1800 gagagcatca actgcgtgca cagggacatt gctgtccgga acatcctggtggcctcccct 1860 gagtgtgtga agctggggga ctttggtctt tcccggtaca ttgaggacgaggactattac 1920 aaagcctctg tgactcgtct ccccatcaaa tggatgtccc cagagtccattaacttccga 1980 cgcttcacga cagccagtga cgtctggatg ttcgccgtgt gcatgtgggagatcctgagc 2040 tttgggaagc agcccttctt ctggctggag aacaaggatg tcatcggggtgctggagaaa 2100 ggagaccggc tgcccaagcc tgatctctgt ccaccggtcc tttataccctcatgacccgc 2160 tgctgggact acgaccccag tgaccggccc cgcttcaccg agctggtgtgcagcctcagt 2220 gacgtttatc agatggagaa ggacattgcc atggagcaag agaggaatgctcgctaccga 2280 acccccaaaa tcttggagcc cacagccttc caggaacccc cacccaagcccagccgacct 2340 aagtacagac cccctccgca aaccaacctc ctggctccaa agctgcagttccaggttcct 2400 gagggtctgt gtgccagctc tcctacgctc accagcccta tggagtatccatctcccgtt 2460 aactcactgc acaccccacc tctccaccgg cacaatgtct tcaaacgccacagcatgggg 2520 gaggaggact tcatccaacc cagcagccga gaagaggccc agcagctgtgggaggctgaa 2580 aaggtcaaaa tgcggcaaat cctggacaaa cagcagaagc agatggtggaggactaccag 2640 tggctcaggc aggaggagaa gtccctggac cccatggttt atatgaatgataagtcccca 2700 ttgacgccag agaaggaggt cggctacctg gagttcacag ggcccccacagaagcccccg 2760 aggctgggcg cacagtccat ccagcccaca gctaacctgg accggaccgatgacctggtg 2820 tacctcaatg tcatggagct ggtgcgggcc gtgctggagc tcaagaatgagctctgtcag 2880 ctgccccccg agggctacgt ggtggtggtg aagaatgtgg ggctgaccctgcggaagctc 2940 atcgggagcg tggatgatct cctgccttcc ttgccgtcat cttcacggacagagatcgag 3000 ggcacccaga aactgctcaa caaagacctg gcagagctca tcaacaagatgcggctggcg 3060 cagcagaacg ccgtgacctc cctgagtgag gagtgcaaga ggcagatgctgacggcttca 3120 cacaccctgg ctgtggacgc caagaacctg ctcgacgctg tggaccaggccaaggttctg 3180 gccaatctgg cccacccacc tgcagagtga cggagggtgg gggccacctgcctgcgtctt 3240 ccgcccctgc ctgccatgta cctcccctgc cttgctgttg gtcatgtgggtcttccaggg 3300 agaaggccaa ggggagtcac cttcccttgc cactttgcac gacgccctctccccacccct 3360 acccctggct gtactgctca ggctgcagct ggacagaggg gactctgggctatggacaca 3420 gggtgacggt gacaaagatg gctcagaggg ggactgctgc tgcctggccactgctcccta 3480 agccagcctg gtccatgcag ggggctcctg ggggtgggga ggtgtcacatggtgccccta 3540 gctttatata tggacatggc aggccgattt gggaaccaag ctattcctttcccttcctct 3600 tctcccctca gatgtccctt gatgcacaga gaagctgggg aggagctttgttttcggggg 3660 tcaggcagcc agtgagatga gggatgggcc tggcattctt gtacagtgtatattgaaatt 3720 tatttaatgt gaggtttggt ctggactgac agcatgtgcc ctcctgagggaggaccaggg 3780 cacagtccag gaacaagcta attgggagtc caggcacagg atgctgtgttgtcaacaaac 3840 caagcatcag ggggaagaag cagagagatg cggccaagat aggaccttgggccaaatccg 3900 ctctcttcct gcccctcttt ctctttcttc ctttactttc ccttgcttttccctcttttc 3960 ttactcctcc tctttctctc ccccaccccc attctcatct gcacccttcttttctcatgt 4020 gtttgcataa acattctttt aacttctttc tatttgactt gtggttgaattaaaattgtc 4080 ccatttgca 4089 8 1009 PRT Homo sapiens calcium dependenttyrosine kinase focal adhesion kinase 2 (FAK2) 8 Met Ser Gly Val Ser GluPro Leu Ser Arg Val Lys Leu Gly Thr Leu 1 5 10 15 Arg Arg Pro Glu GlyPro Ala Glu Pro Met Val Val Val Pro Val Asp 20 25 30 Val Glu Lys Glu AspVal Arg Ile Leu Lys Val Cys Phe Tyr Ser Asn 35 40 45 Ser Phe Asn Pro GlyLys Asn Phe Lys Leu Val Lys Cys Thr Val Gln 50 55 60 Thr Glu Ile Arg GluIle Ile Thr Ser Ile Leu Leu Ser Gly Arg Ile 65 70 75 80 Gly Pro Asn IleArg Leu Ala Glu Cys Tyr Gly Leu Arg Leu Lys His 85 90 95 Met Lys Ser AspGlu Ile His Trp Leu His Pro Gln Met Thr Val Gly 100 105 110 Glu Val GlnAsp Lys Tyr Glu Cys Leu His Val Glu Ala Glu Trp Arg 115 120 125 Tyr AspLeu Gln Ile Arg Tyr Leu Pro Glu Asp Phe Met Glu Ser Leu 130 135 140 LysGlu Asp Arg Thr Thr Leu Leu Tyr Phe Tyr Gln Gln Leu Arg Asn 145 150 155160 Asp Tyr Met Gln Arg Tyr Ala Ser Lys Val Ser Glu Gly Met Ala Leu 165170 175 Gln Leu Gly Cys Leu Glu Leu Arg Arg Phe Phe Lys Asp Met Pro His180 185 190 Asn Ala Leu Asp Lys Lys Ser Asn Phe Glu Leu Leu Glu Lys GluVal 195 200 205 Gly Leu Asp Leu Phe Phe Pro Lys Gln Met Gln Glu Asn LeuLys Pro 210 215 220 Lys Gln Phe Arg Lys Met Ile Gln Gln Thr Phe Gln GlnTyr Ala Ser 225 230 235 240 Leu Arg Glu Glu Glu Cys Val Met Lys Phe PheAsn Thr Leu Ala Gly 245 250 255 Phe Ala Asn Ile Asp Gln Glu Thr Tyr ArgCys Glu Leu Ile Gln Gly 260 265 270 Trp Asn Ile Thr Val Asp Leu Val IleGly Pro Lys Gly Ile Arg Gln 275 280 285 Leu Thr Ser Gln Asp Ala Lys ProThr Cys Leu Ala Glu Phe Lys Gln 290 295 300 Ile Arg Ser Ile Arg Cys LeuPro Leu Glu Glu Gly Gln Ala Val Leu 305 310 315 320 Gln Leu Gly Ile GluGly Ala Pro Gln Ala Leu Ser Ile Lys Thr Ser 325 330 335 Ser Leu Ala GluAla Glu Asn Met Ala Asp Leu Ile Asp Gly Tyr Cys 340 345 350 Arg Leu GlnGly Glu His Gln Gly Ser Leu Ile Ile His Pro Arg Lys 355 360 365 Asp GlyGlu Lys Arg Asn Ser Leu Pro Gln Ile Pro Met Leu Asn Leu 370 375 380 GluAla Arg Arg Ser His Leu Ser Glu Ser Cys Ser Ile Glu Ser Asp 385 390 395400 Ile Tyr Ala Glu Ile Pro Asp Glu Thr Leu Arg Arg Pro Gly Gly Pro 405410 415 Gln Tyr Gly Ile Ala Arg Glu Asp Val Val Leu Asn Arg Ile Leu Gly420 425 430 Glu Gly Phe Phe Gly Glu Val Tyr Glu Gly Val Tyr Thr Asn HisLys 435 440 445 Gly Glu Lys Ile Asn Val Ala Val Lys Thr Cys Lys Lys AspCys Thr 450 455 460 Leu Asp Asn Lys Glu Lys Phe Met Ser Glu Ala Val IleMet Lys Asn 465 470 475 480 Leu Asp His Pro His Ile Val Lys Leu Ile GlyIle Ile Glu Glu Glu 485 490 495 Pro Thr Trp Ile Ile Met Glu Leu Tyr ProTyr Gly Glu Leu Gly His 500 505 510 Tyr Leu Glu Arg Asn Lys Asn Ser LeuLys Val Leu Thr Leu Val Leu 515 520 525 Tyr Ser Leu Gln Ile Cys Lys AlaMet Ala Tyr Leu Glu Ser Ile Asn 530 535 540 Cys Val His Arg Asp Ile AlaVal Arg Asn Ile Leu Val Ala Ser Pro 545 550 555 560 Glu Cys Val Lys LeuGly Asp Phe Gly Leu Ser Arg Tyr Ile Glu Asp 565 570 575 Glu Asp Tyr TyrLys Ala Ser Val Thr Arg Leu Pro Ile Lys Trp Met 580 585 590 Ser Pro GluSer Ile Asn Phe Arg Arg Phe Thr Thr Ala Ser Asp Val 595 600 605 Trp MetPhe Ala Val Cys Met Trp Glu Ile Leu Ser Phe Gly Lys Gln 610 615 620 ProPhe Phe Trp Leu Glu Asn Lys Asp Val Ile Gly Val Leu Glu Lys 625 630 635640 Gly Asp Arg Leu Pro Lys Pro Asp Leu Cys Pro Pro Val Leu Tyr Thr 645650 655 Leu Met Thr Arg Cys Trp Asp Tyr Asp Pro Ser Asp Arg Pro Arg Phe660 665 670 Thr Glu Leu Val Cys Ser Leu Ser Asp Val Tyr Gln Met Glu LysAsp 675 680 685 Ile Ala Met Glu Gln Glu Arg Asn Ala Arg Tyr Arg Thr ProLys Ile 690 695 700 Leu Glu Pro Thr Ala Phe Gln Glu Pro Pro Pro Lys ProSer Arg Pro 705 710 715 720 Lys Tyr Arg Pro Pro Pro Gln Thr Asn Leu LeuAla Pro Lys Leu Gln 725 730 735 Phe Gln Val Pro Glu Gly Leu Cys Ala SerSer Pro Thr Leu Thr Ser 740 745 750 Pro Met Glu Tyr Pro Ser Pro Val AsnSer Leu His Thr Pro Pro Leu 755 760 765 His Arg His Asn Val Phe Lys ArgHis Ser Met Arg Glu Glu Asp Phe 770 775 780 Ile Gln Pro Ser Ser Arg GluGlu Ala Gln Gln Leu Trp Glu Ala Glu 785 790 795 800 Lys Val Lys Met ArgGln Ile Leu Asp Lys Gln Gln Lys Gln Met Val 805 810 815 Glu Asp Tyr GlnTrp Leu Arg Gln Glu Glu Lys Ser Leu Asp Pro Met 820 825 830 Val Tyr MetAsn Asp Lys Ser Pro Leu Thr Pro Glu Lys Glu Val Gly 835 840 845 Tyr LeuGlu Phe Thr Gly Pro Pro Gln Lys Pro Pro Arg Leu Gly Ala 850 855 860 GlnSer Ile Gln Pro Thr Ala Asn Leu Asp Arg Thr Asp Asp Leu Val 865 870 875880 Tyr Leu Asn Val Met Glu Leu Val Arg Ala Val Leu Glu Leu Lys Asn 885890 895 Glu Leu Cys Gln Leu Pro Pro Glu Gly Tyr Val Val Val Val Lys Asn900 905 910 Val Gly Leu Thr Leu Arg Lys Leu Ile Gly Ser Val Asp Asp LeuLeu 915 920 925 Pro Ser Leu Pro Ser Ser Ser Arg Thr Glu Ile Glu Gly ThrGln Lys 930 935 940 Leu Leu Asn Lys Asp Leu Ala Glu Leu Ile Asn Lys MetArg Leu Ala 945 950 955 960 Gln Gln Asn Ala Val Thr Ser Leu Ser Glu GluCys Lys Arg Gln Met 965 970 975 Leu Thr Ala Ser His Thr Leu Ala Val AspAla Lys Asn Leu Leu Asp 980 985 990 Ala Val Asp Gln Ala Lys Val Leu AlaAsn Leu Ala His Pro Pro Ala 995 1000 1005 Glu 9 2195 DNA Homo sapiensserine threonine protein kinase casein kinase 2, alpha 1 subunit isoforma, transcript variant 2 (CK2, CK2alpha), CK2 catalytic subunit alpha 9aggggagagc ggccgccgcc gctgccgctt ccaccacagt ttgaagaaaa caggtctgaa 60acaaggtctt acccccagct gcttctgaac acagtgactg ccagatctcc aaacatcaag 120tccagctttg tccgccaacc tgtctgacat gtcgggaccc gtgccaagca gggccagagt 180ttacacagat gttaatacac acagacctcg agaatactgg gattacgagt cacatgtggt 240ggaatgggga aatcaagatg actaccagct ggttcgaaaa ttaggccgag gtaaatacag 300tgaagtattt gaagccatca acatcacaaa taatgaaaaa gttgttgtta aaattctcaa 360gccagtaaaa aagaagaaaa ttaagcgtga aataaagatt ttggagaatt tgagaggagg 420tcccaacatc atcacactgg cagacattgt aaaagaccct gtgtcacgaa cccccgcctt 480ggtttttgaa cacgtaaaca acacagactt caagcaattg taccagacgt taacagacta 540tgatattcga ttttacatgt atgagattct gaaggccctg gattattgtc acagcatggg 600aattatgcac agagatgtca agccccataa tgtcatgatt gatcatgagc acagaaagct 660acgactaata gactggggtt tggctgagtt ttatcatcct ggccaagaat ataatgtccg 720agttgcttcc cgatacttca aaggtcctga gctacttgta gactatcaga tgtacgatta 780tagtttggat atgtggagtt tgggttgtat gctggcaagt atgatctttc ggaaggagcc 840atttttccat ggacatgaca attatgatca gttggtgagg atagccaagg ttctggggac 900agaagattta tatgactata ttgacaaata caacattgaa ttagatccac gtttcaatga 960tatcttgggc agacactctc gaaagcgatg ggaacgcttt gtccacagtg aaaatcagca 1020ccttgtcagc cctgaggcct tggatttcct ggacaaactg ctgcgatatg accaccagtc 1080acggcttact gcaagagagg caatggagca cccctatttc tacactgttg tgaaggacca 1140ggctcgaatg ggttcatcta gcatgccagg gggcagtacg cccgtcagca gcgccaatat 1200gatgtcaggg atttcttcag tgccaacccc ttcacccctt ggacctctgg caggctcacc 1260agtgattgct gctgccaacc cccttgggat gcctgttcca gctgccgctg gcgctcagca 1320gtaacggccc tatctgtctc ctgatgcctg agcagaggtg ggggagtcca ccctctcctt 1380gatgcagctt gcgcctggcg gggaggggtg aaacacttca gaagcaccgt gtctgaaccg 1440ttgcttgtgg atttatagta gttcagtcat aaaaaaaaaa ttataatagg ctgattttct 1500tttttctttt tttttttaac tcgaactttt cataactcag gggattccct gaaaaattac 1560ctgcaggtgg aatatttcat ggacaaattt ttttttctcc cctcccaaat ttagttcctc 1620atcacaaaag aacaaagata aaccagcctc aatcccggct gctgcattta ggtggagact 1680tcttcccatt cccaccattg ttcctccacc gtcccacact ttagggggtt ggtatctcgt 1740gctcttctcc agagattaca aaaatgtagc ttctcagggg aggcaggaag aaaggaagga 1800aggaaagaag gaagggagga cccaatctat aggagcagtg gactgcttgc tggtcgctta 1860catcacttta ctccataagc gcttcagtgg ggttatccta gtggctcttg tggaagtgtg 1920tcttagttac atcaagatgt tgaaaatcta cccaaaatgc agacagatac taaaaacttc 1980tgttcagtaa gaatcatgtc ttactgatct aaccctaaat ccaactcatt tatactttta 2040tttttagttc agtttaaaat gttgatacct tccctcccag gctccttacc ttggtctttt 2100ccctgttcat ctcccaacat gctgtgctcc atagctggta ggagagggaa ggcaaaatct 2160ttcttagttt tctttgtctt ggccattttg aattc 2195 10 391 PRT Homo sapiensserine threonine protein kinase casein kinase 2, alpha 1 subunit isoforma, transcript variant 2 (CK2, CK2alpha), CK2 catalytic subunit alpha 10Met Ser Gly Pro Val Pro Ser Arg Ala Arg Val Tyr Thr Asp Val Asn 1 5 1015 Thr His Arg Pro Arg Glu Tyr Trp Asp Tyr Glu Ser His Val Val Glu 20 2530 Trp Gly Asn Gln Asp Asp Tyr Gln Leu Val Arg Lys Leu Gly Arg Gly 35 4045 Lys Tyr Ser Glu Val Phe Glu Ala Ile Asn Ile Thr Asn Asn Glu Lys 50 5560 Val Val Val Lys Ile Leu Lys Pro Val Lys Lys Lys Lys Ile Lys Arg 65 7075 80 Glu Ile Lys Ile Leu Glu Asn Leu Arg Gly Gly Pro Asn Ile Ile Thr 8590 95 Leu Ala Asp Ile Val Lys Asp Pro Val Ser Arg Thr Pro Ala Leu Val100 105 110 Phe Glu His Val Asn Asn Thr Asp Phe Lys Gln Leu Tyr Gln ThrLeu 115 120 125 Thr Asp Tyr Asp Ile Arg Phe Tyr Met Tyr Glu Ile Leu LysAla Leu 130 135 140 Asp Tyr Cys His Ser Met Gly Ile Met His Arg Asp ValLys Pro His 145 150 155 160 Asn Val Met Ile Asp His Glu His Arg Lys LeuArg Leu Ile Asp Trp 165 170 175 Gly Leu Ala Glu Phe Tyr His Pro Gly GlnGlu Tyr Asn Val Arg Val 180 185 190 Ala Ser Arg Tyr Phe Lys Gly Pro GluLeu Leu Val Asp Tyr Gln Met 195 200 205 Tyr Asp Tyr Ser Leu Asp Met TrpSer Leu Gly Cys Met Leu Ala Ser 210 215 220 Met Ile Phe Arg Lys Glu ProPhe Phe His Gly His Asp Asn Tyr Asp 225 230 235 240 Gln Leu Val Arg IleAla Lys Val Leu Gly Thr Glu Asp Leu Tyr Asp 245 250 255 Tyr Ile Asp LysTyr Asn Ile Glu Leu Asp Pro Arg Phe Asn Asp Ile 260 265 270 Leu Gly ArgHis Ser Arg Lys Arg Trp Glu Arg Phe Val His Ser Glu 275 280 285 Asn GlnHis Leu Val Ser Pro Glu Ala Leu Asp Phe Leu Asp Lys Leu 290 295 300 LeuArg Tyr Asp His Gln Ser Arg Leu Thr Ala Arg Glu Ala Met Glu 305 310 315320 His Pro Tyr Phe Tyr Thr Val Val Lys Asp Gln Ala Arg Met Gly Ser 325330 335 Ser Ser Met Pro Gly Gly Ser Thr Pro Val Ser Ser Ala Asn Met Met340 345 350 Ser Gly Ile Ser Ser Val Pro Thr Pro Ser Pro Leu Gly Pro LeuAla 355 360 365 Gly Ser Pro Val Ile Ala Ala Ala Asn Pro Leu Gly Met ProVal Pro 370 375 380 Ala Ala Ala Gly Ala Gln Gln 385 390 11 4626 DNA Homosapiens cMET proto-oncogene tyrosine kinase 11 gaattccgcc ctcgccgcccgcggcgcccc gagcgctttg tgagcagatg cggagccgag 60 tggagggcgc gagccagatgcggggcgaca gctgacttgc tgagaggagg cggggaggcg 120 cggagcgcgc gtgtggtccttgcgccgctg acttctccac tggttcctgg gcaccgaaag 180 ataaacctct cataatgaaggcccccgctg tgcttgcacc tggcatcctc gtgctcctgt 240 ttaccttggt gcagaggagcaatggggagt gtaaagaggc actagcaaag tccgagatga 300 atgtgaatat gaagtatcagcttcccaact tcaccgcgga aacacccatc cagaatgtca 360 ttctacatga gcatcacattttccttggtg ccactaacta catttatgtt ttaaatgagg 420 aagaccttca gaaggttgctgagtacaaga ctgggcctgt gctggaacac ccagattgtt 480 tcccatgtca ggactgcagcagcaaagcca atttatcagg aggtgtttgg aaagataaca 540 tcaacatggc tctagttgtcgacacctact atgatgatca actcattagc tgtggcagcg 600 tcaacagagg gacctgccagcgacatgtct ttccccacaa tcatactgct gacatacagt 660 cggaggttca ctgcatattctccccacaga tagaagagcc cagccagtgt cctgactgtg 720 tggtgagcgc cctgggagccaaagtccttt catctgtaaa ggaccggttc atcaacttct 780 ttgtaggcaa taccataaattcttcttatt tcccagatca tccattgcat tcgatatcag 840 tgagaaggct aaaggaaacgaaagatggtt ttatgttttt gacggaccag tcctacattg 900 atgttttacc tgagttcagagattcttacc ccattaagta tgtccatgcc tttgaaagca 960 acaattttat ttacttcttgacggtccaaa gggaaactct agatgctcag acttttcaca 1020 caagaataat caggttctgttccataaact ctggattgca ttcctacatg gaaatgcctc 1080 tggagtgtat tctcacagaaaagagaaaaa agagatccac aaagaaggaa gtgtttaata 1140 tacttcaggc tgcgtatgtcagcaagcctg gggcccagct tgctagacaa ataggagcca 1200 gcctgaatga tgacattcttttcggggtgt tcgcacaaag caagccagat tctgccgaac 1260 caatggatcg atctgccatgtgtgcattcc ctatcaaata tgtcaacgac ttcttcaaca 1320 agatcgtcaa caaaaacaatgtgagatgtc tccagcattt ttacggaccc aatcatgagc 1380 actgctttaa taggacacttctgagaaatt catcaggctg tgaagcgcgc cgtgatgaat 1440 atcgaacaga gtttaccacagctttgcagc gcgttgactt attcatgggt caattcagcg 1500 aagtcctctt aacatctatatccaccttca ttaaaggaga cctcaccata gctaatcttg 1560 ggacatcaga gggtcgcttcatgcaggttg tggtttctcg atcaggacca tcaacccctc 1620 atgtgaattt tctcctggactcccatccag tgtctccaga agtgattgtg gagcatacat 1680 taaaccaaaa tggctacacactggttatca ctgggaagaa gatcacgaag atcccattga 1740 atggcttggg ctgcagacatttccagtcct gcagtcaatg cctctctgcc ccaccctttg 1800 ttcagtgtgg ctggtgccacgacaaatgtg tgcgatcgga ggaatgcctg agcgggacat 1860 ggactcaaca gatctgtctgcctgcaatct acaaggtttt cccaaatagt gcaccccttg 1920 aaggagggac aaggctgaccatatgtggct gggactttgg atttcggagg aataataaat 1980 ttgatttaaa gaaaactagagttctccttg gaaatgagag ctgcaccttg actttaagtg 2040 agagcacgat gaatacattgaaatgcacag ttggtcctgc catgaataag catttcaata 2100 tgtccataat tatttcaaatggccacggga caacacaata cagtacattc tcctatgtgg 2160 atcctgtaat aacaagtatttcgccgaaat acggtcctat ggctggtggc actttactta 2220 ctttaactgg aaattacctaaacagtggga attctagaca catttcaatt ggtggaaaaa 2280 catgtacttt aaaaagtgtgtcaaacagta ttcttgaatg ttatacccca gcccaaacca 2340 tttcaactga gtttgctgttaaattgaaaa ttgacttagc caaccgagag acaagcatct 2400 tcagttaccg tgaagatcccattgtctatg aaattcatcc aaccaaatct tttattagta 2460 cttggtggaa agaacctctcaacattgtca gttttctatt ttgctttgcc agtggtggga 2520 gcacaataac aggtgttgggaaaaacctga attcagttag tgtcccgaga atggtcataa 2580 atgtgcatga agcaggaaggaactttacag tggcatgtca acatcgctct aattcagaga 2640 taatctgttg taccactccttccctgcaac agctgaatct gcaactcccc ctgaaaacca 2700 aagccttttt catgttagatgggatccttt ccaaatactt tgatctcatt tatgtacata 2760 atcctgtgtt taagccttttgaaaagccag tgatgatctc aatgggcaat gaaaatgtac 2820 tggaaattaa gggaaatgatattgaccctg aagcagttaa aggtgaagtg ttaaaagttg 2880 gaaataagag ctgtgagaatatacacttac attctgaagc cgttttatgc acggtcccca 2940 atgacctgct gaaattgaacagcgagctaa atatagagtg gaagcaagca atttcttcaa 3000 ccgtccttgg aaaagtaatagttcaaccag atcagaattt cacaggattg attgctggtg 3060 ttgtctcaat atcaacagcactgttattac tacttgggtt tttcctgtgg ctgaaaaaga 3120 gaaagcaaat taaagatctgggcagtgaat tagttcgcta cgatgcaaga gtacacactc 3180 ctcatttgga taggcttgtaagtgcccgaa gtgtaagccc aactacagaa atggtttcaa 3240 atgaatctgt agactaccgagctacttttc cagaagatca gtttcctaat tcatctcaga 3300 acggttcatg ccgacaagtgcagtatcctc tgacagacat gtcccccatc ctaactagtg 3360 gggactctga tatatccagtccattactgc aaaatactgt ccacattgac ctcagtgctc 3420 taaatccaga gctggtccaggcagtgcagc atgtagtgat tgggcccagt agcctgattg 3480 tgcatttcaa tgaagtcataggaagagggc attttggttg tgtatatcat gggactttgt 3540 tggacaatga tggcaagaaaattcactgtg ctgtgaaatc cttgaacaga atcactgaca 3600 taggagaagt ttcccaatttctgaccgagg gaatcatcat gaaagatttt agtcatccca 3660 atgtcctctc gctcctgggaatctgcctgc gaagtgaagg gtctccgctg gtggtcctac 3720 catacatgaa acatggagatcttcgaaatt tcattcgaaa tgagactcat aatccaactg 3780 taaaagatct tattggctttggtcttcaag tagccaaagc gatgaaatat cttgcaagca 3840 aaaagtttgt ccacagagacttggctgcaa gaaactgtat gctggatgaa aaattcacag 3900 tcaaggttgc tgattttggtcttgccagag acatgtatga taaagaatac tatagtgtac 3960 acaacaaaac aggtgcaaagctgccagtga agtggatggc tttggaaagt ctgcaaactc 4020 aaaagtttac caccaagtcagatgtgtggt cctttggcgt cgtcctctgg gagctgatga 4080 caagaggagc cccaccttatcctgacgtaa acacctttga tataactgtt tacttgttgc 4140 aagggagaag actcctacaacccgaatact gcccagaccc cttatatgaa gtaatgctaa 4200 aatgctggca ccctaaagccgaaatgcgcc catccttttc tgaactggtg tcccggatat 4260 cagcgatctt ctctactttcattggggagc actatgtcca tgtgaacgct acttatgtga 4320 acgtaaaatg tgtcgctccgtatccttctc tgttgtcatc agaagataac gctgatgatg 4380 aggtggacac acgaccagcctccttctggg agacatcata gtgctagtac tatgtcaaag 4440 caacagtcca cactttgtccaatggttttt tcactgcctg acctttaaaa ggccatcgat 4500 attctttgct ccttgccataggacttgtat tgttatttaa attactggat tctaaggaat 4560 ttcttatctg acagagcatcagaaccagag gcttggtccc acaggccagg gaccaatgcg 4620 ctgcag 4626 12 1408 PRTHomo sapiens cMET proto-oncogene tyrosine kinase 12 Met Lys Ala Pro AlaVal Leu Ala Pro Gly Ile Leu Val Leu Leu Phe 1 5 10 15 Thr Leu Val GlnArg Ser Asn Gly Glu Cys Lys Glu Ala Leu Ala Lys 20 25 30 Ser Glu Met AsnVal Asn Met Lys Tyr Gln Leu Pro Asn Phe Thr Ala 35 40 45 Glu Thr Pro IleGln Asn Val Ile Leu His Glu His His Ile Phe Leu 50 55 60 Gly Ala Thr AsnTyr Ile Tyr Val Leu Asn Glu Glu Asp Leu Gln Lys 65 70 75 80 Val Ala GluTyr Lys Thr Gly Pro Val Leu Glu His Pro Asp Cys Phe 85 90 95 Pro Cys GlnAsp Cys Ser Ser Lys Ala Asn Leu Ser Gly Gly Val Trp 100 105 110 Lys AspAsn Ile Asn Met Ala Leu Val Val Asp Thr Tyr Tyr Asp Asp 115 120 125 GlnLeu Ile Ser Cys Gly Ser Val Asn Arg Gly Thr Cys Gln Arg His 130 135 140Val Phe Pro His Asn His Thr Ala Asp Ile Gln Ser Glu Val His Cys 145 150155 160 Ile Phe Ser Pro Gln Ile Glu Glu Pro Ser Gln Cys Pro Asp Cys Val165 170 175 Val Ser Ala Leu Gly Ala Lys Val Leu Ser Ser Val Lys Asp ArgPhe 180 185 190 Ile Asn Phe Phe Val Gly Asn Thr Ile Asn Ser Ser Tyr PhePro Asp 195 200 205 His Pro Leu His Ser Ile Ser Val Arg Arg Leu Lys GluThr Lys Asp 210 215 220 Gly Phe Met Phe Leu Thr Asp Gln Ser Tyr Ile AspVal Leu Pro Glu 225 230 235 240 Phe Arg Asp Ser Tyr Pro Ile Lys Tyr ValHis Ala Phe Glu Ser Asn 245 250 255 Asn Phe Ile Tyr Phe Leu Thr Val GlnArg Glu Thr Leu Asp Ala Gln 260 265 270 Thr Phe His Thr Arg Ile Ile ArgPhe Cys Ser Ile Asn Ser Gly Leu 275 280 285 His Ser Tyr Met Glu Met ProLeu Glu Cys Ile Leu Thr Glu Lys Arg 290 295 300 Lys Lys Arg Ser Thr LysLys Glu Val Phe Asn Ile Leu Gln Ala Ala 305 310 315 320 Tyr Val Ser LysPro Gly Ala Gln Leu Ala Arg Gln Ile Gly Ala Ser 325 330 335 Leu Asn AspAsp Ile Leu Phe Gly Val Phe Ala Gln Ser Lys Pro Asp 340 345 350 Ser AlaGlu Pro Met Asp Arg Ser Ala Met Cys Ala Phe Pro Ile Lys 355 360 365 TyrVal Asn Asp Phe Phe Asn Lys Ile Val Asn Lys Asn Asn Val Arg 370 375 380Cys Leu Gln His Phe Tyr Gly Pro Asn His Glu His Cys Phe Asn Arg 385 390395 400 Thr Leu Leu Arg Asn Ser Ser Gly Cys Glu Ala Arg Arg Asp Glu Tyr405 410 415 Arg Thr Glu Phe Thr Thr Ala Leu Gln Arg Val Asp Leu Phe MetGly 420 425 430 Gln Phe Ser Glu Val Leu Leu Thr Ser Ile Ser Thr Phe IleLys Gly 435 440 445 Asp Leu Thr Ile Ala Asn Leu Gly Thr Ser Glu Gly ArgPhe Met Gln 450 455 460 Val Val Val Ser Arg Ser Gly Pro Ser Thr Pro HisVal Asn Phe Leu 465 470 475 480 Leu Asp Ser His Pro Val Ser Pro Glu ValIle Val Glu His Thr Leu 485 490 495 Asn Gln Asn Gly Tyr Thr Leu Val IleThr Gly Lys Lys Ile Thr Lys 500 505 510 Ile Pro Leu Asn Gly Leu Gly CysArg His Phe Gln Ser Cys Ser Gln 515 520 525 Cys Leu Ser Ala Pro Pro PheVal Gln Cys Gly Trp Cys His Asp Lys 530 535 540 Cys Val Arg Ser Glu GluCys Leu Ser Gly Thr Trp Thr Gln Gln Ile 545 550 555 560 Cys Leu Pro AlaIle Tyr Lys Val Phe Pro Asn Ser Ala Pro Leu Glu 565 570 575 Gly Gly ThrArg Leu Thr Ile Cys Gly Trp Asp Phe Gly Phe Arg Arg 580 585 590 Asn AsnLys Phe Asp Leu Lys Lys Thr Arg Val Leu Leu Gly Asn Glu 595 600 605 SerCys Thr Leu Thr Leu Ser Glu Ser Thr Met Asn Thr Leu Lys Cys 610 615 620Thr Val Gly Pro Ala Met Asn Lys His Phe Asn Met Ser Ile Ile Ile 625 630635 640 Ser Asn Gly His Gly Thr Thr Gln Tyr Ser Thr Phe Ser Tyr Val Asp645 650 655 Pro Val Ile Thr Ser Ile Ser Pro Lys Tyr Gly Pro Met Ala GlyGly 660 665 670 Thr Leu Leu Thr Leu Thr Gly Asn Tyr Leu Asn Ser Gly AsnSer Arg 675 680 685 His Ile Ser Ile Gly Gly Lys Thr Cys Thr Leu Lys SerVal Ser Asn 690 695 700 Ser Ile Leu Glu Cys Tyr Thr Pro Ala Gln Thr IleSer Thr Glu Phe 705 710 715 720 Ala Val Lys Leu Lys Ile Asp Leu Ala AsnArg Glu Thr Ser Ile Phe 725 730 735 Ser Tyr Arg Glu Asp Pro Ile Val TyrGlu Ile His Pro Thr Lys Ser 740 745 750 Phe Ile Ser Thr Trp Trp Lys GluPro Leu Asn Ile Val Ser Phe Leu 755 760 765 Phe Cys Phe Ala Ser Gly GlySer Thr Ile Thr Gly Val Gly Lys Asn 770 775 780 Leu Asn Ser Val Ser ValPro Arg Met Val Ile Asn Val His Glu Ala 785 790 795 800 Gly Arg Asn PheThr Val Ala Cys Gln His Arg Ser Asn Ser Glu Ile 805 810 815 Ile Cys CysThr Thr Pro Ser Leu Gln Gln Leu Asn Leu Gln Leu Pro 820 825 830 Leu LysThr Lys Ala Phe Phe Met Leu Asp Gly Ile Leu Ser Lys Tyr 835 840 845 PheAsp Leu Ile Tyr Val His Asn Pro Val Phe Lys Pro Phe Glu Lys 850 855 860Pro Val Met Ile Ser Met Gly Asn Glu Asn Val Leu Glu Ile Lys Gly 865 870875 880 Asn Asp Ile Asp Pro Glu Ala Val Lys Gly Glu Val Leu Lys Val Gly885 890 895 Asn Lys Ser Cys Glu Asn Ile His Leu His Ser Glu Ala Val LeuCys 900 905 910 Thr Val Pro Asn Asp Leu Leu Lys Leu Asn Ser Glu Leu AsnIle Glu 915 920 925 Trp Lys Gln Ala Ile Ser Ser Thr Val Leu Gly Lys ValIle Val Gln 930 935 940 Pro Asp Gln Asn Phe Thr Gly Leu Ile Ala Gly ValVal Ser Ile Ser 945 950 955 960 Thr Ala Leu Leu Leu Leu Leu Gly Phe PheLeu Trp Leu Lys Lys Arg 965 970 975 Lys Gln Ile Lys Asp Leu Gly Ser GluLeu Val Arg Tyr Asp Ala Arg 980 985 990 Val His Thr Pro His Leu Asp ArgLeu Val Ser Ala Arg Ser Val Ser 995 1000 1005 Pro Thr Thr Glu Met ValSer Asn Glu Ser Val Asp Tyr Arg Ala Thr 1010 1015 1020 Phe Pro Glu AspGln Phe Pro Asn Ser Ser Gln Asn Gly Ser Cys Arg 1025 1030 1035 1040 GlnVal Gln Tyr Pro Leu Thr Asp Met Ser Pro Ile Leu Thr Ser Gly 1045 10501055 Asp Ser Asp Ile Ser Ser Pro Leu Leu Gln Asn Thr Val His Ile Asp1060 1065 1070 Leu Ser Ala Leu Asn Pro Glu Leu Val Gln Ala Val Gln HisVal Val 1075 1080 1085 Ile Gly Pro Ser Ser Leu Ile Val His Phe Asn GluVal Ile Gly Arg 1090 1095 1100 Gly His Phe Gly Cys Val Tyr His Gly ThrLeu Leu Asp Asn Asp Gly 1105 1110 1115 1120 Lys Lys Ile His Cys Ala ValLys Ser Leu Asn Arg Ile Thr Asp Ile 1125 1130 1135 Gly Glu Val Ser GlnPhe Leu Thr Glu Gly Ile Ile Met Lys Asp Phe 1140 1145 1150 Ser His ProAsn Val Leu Ser Leu Leu Gly Ile Cys Leu Arg Ser Glu 1155 1160 1165 GlySer Pro Leu Val Val Leu Pro Tyr Met Lys His Gly Asp Leu Arg 1170 11751180 Asn Phe Ile Arg Asn Glu Thr His Asn Pro Thr Val Lys Asp Leu Ile1185 1190 1195 1200 Gly Phe Gly Leu Gln Val Ala Lys Ala Met Lys Tyr LeuAla Ser Lys 1205 1210 1215 Lys Phe Val His Arg Asp Leu Ala Ala Arg AsnCys Met Leu Asp Glu 1220 1225 1230 Lys Phe Thr Val Lys Val Ala Asp PheGly Leu Ala Arg Asp Met Tyr 1235 1240 1245 Asp Lys Glu Tyr Tyr Ser ValHis Asn Lys Thr Gly Ala Lys Leu Pro 1250 1255 1260 Val Lys Trp Met AlaLeu Glu Ser Leu Gln Thr Gln Lys Phe Thr Thr 1265 1270 1275 1280 Lys SerAsp Val Trp Ser Phe Gly Val Val Leu Trp Glu Leu Met Thr 1285 1290 1295Arg Gly Ala Pro Pro Tyr Pro Asp Val Asn Thr Phe Asp Ile Thr Val 13001305 1310 Tyr Leu Leu Gln Gly Arg Arg Leu Leu Gln Pro Glu Tyr Cys ProAsp 1315 1320 1325 Pro Leu Tyr Glu Val Met Leu Lys Cys Trp His Pro LysAla Glu Met 1330 1335 1340 Arg Pro Ser Phe Ser Glu Leu Val Ser Arg IleSer Ala Ile Phe Ser 1345 1350 1355 1360 Thr Phe Ile Gly Glu His Tyr ValHis Val Asn Ala Thr Tyr Val Asn 1365 1370 1375 Val Lys Cys Val Ala ProTyr Pro Ser Leu Leu Ser Ser Glu Asp Asn 1380 1385 1390 Ala Asp Asp GluVal Asp Thr Arg Pro Ala Ser Phe Trp Glu Thr Ser 1395 1400 1405 13 3350DNA Homo sapiens flap structure-specific endonuclease 1 (FEN1) 5′-3′exonuclease 13 cacagtccac tctgtcaggg tttaaggcag gaaaaacatg ctcattttgatggtaatatt 60 cttccttctc aacattccat ttctcctggc aaatttcatg gatcccagatgcttttggaa 120 aataaatttg aatgaaatca aggatgaagt ccttgggatg acttgttccttcatccttga 180 aacagttcag aagactatgg acaaagatta tttcaaccag actctgaatgtcctaaatac 240 aactacaaac cacaaatatg ccttggcatt ggcctttaca gtggatgaaatcaacaggaa 300 tcctgatctt ttaccaaata tgtctctgat tataaaatac aatttgggtcattgtgatgg 360 aaaaactgta acaactctat ccgatttatt taatccaaat aatcatctccatttccccaa 420 ttatttatgt aatgaaggga ttatgtgttt ggttctgctt acaggaccacattggagagc 480 atctttatat ctctggatat ccgtgtatgt ctacctgtct ccacatttccttcagctttc 540 ctatggacct ttctactcca tcttcagtga taatgaacaa tatccttatctctatcagat 600 gggcccaaag gactcatcac tagcattggc aatggtctcc ttcataatttacttcaagtg 660 gaactgggtt gggctattta tctcagatga tgatcaaggc aatcaatttctctcagagtt 720 gaaaaaagag agccaaacca aggatatttg ctttgccttt gtgaacatgatatcagtcag 780 tgatgtttca tactatcata aaactgaaat gtactacaac caaattgtgatgtcatccac 840 aaaggttatt atcatttatg gggaaacaaa cagtattatt gaattgagcttcagaatgtg 900 gtcatctcca gttaaacaga gaatatgggt caccacaaaa caatttgattgccctaccag 960 taagagagac ttaactcatg gcacattcta tgggaccctt acatttctacaccactatgg 1020 tgagatttct ggctttaaaa attttgtaca gacacggtac aatctcagaagcacagattt 1080 atatctagta atgccagagt ggaaatattt taactatgaa gcctcagcatctaactgtaa 1140 aatactgaga aactatttat ccaatatctc actggaatgg ctaatggaacagaaatttga 1200 catgtcattt agtgattata gtcacaacat atacaatgct gtatatgccattgctcatgc 1260 actccatgag aagaatctgc aagaagttga aaatcaggca ataaacaatgcgaaaggaga 1320 aaatactcac tgcttgaagc taaactcatt tctgagaaag acccacttcactaattctct 1380 tgggaacaga gtaattatga aacagagaga agtagtgcat ggagactataatattgttca 1440 catgtggaat ttctcacaac gccttgggat taaggtgaag ataggacaattcagcccaca 1500 ttttccacag ggtcaacagt tacacttata tgtagacatg actgagttggctacaggaag 1560 tagaaagatg ccatcctcag tgtgcagtgc agattgccat cctggattcagaagaatctg 1620 gaaggaggaa atggcagcct gctgttttgt ttgcaacccc tgccctgaaaatgaaatttc 1680 taatgagacg atggtggtat tttgggtctt cgtgaagcac catgacactcctattgtgaa 1740 ggccaataac agaatcctca gctacctatt aatcgtgtca ctcatgttctgttttctgtg 1800 ctcctttttc ttcattggct atcctaacag agcaacctgt atcttacagcaaatcacatt 1860 tggaatcttc tttactgtgg ctatttccac agttctggcc aaaacaatcactgtggttct 1920 ggctttcaaa gtcacagacc caggaagaca attaagaatc tttttggtatcggggacacc 1980 caactacatt attcccatat gttccctatt gcaatgtatt ctgtgtgcaatctggctagc 2040 agtttctcct ccctttgttg atattgatga acactctgag catggccacatcatcattgt 2100 gtgcaacaag ggctccatta ctgcattcta ctgtgtcctg ggatacttggcctgcctggc 2160 ctttggaagc ttcactatag ctttcttggc aaagaacctg cctgacacattcaacgaagc 2220 caagttcttg accttcagca tgctagtgtt ctgcgctgtc tgggtcaccttcctccctgt 2280 ctaccatagc accaagggca aggtcatggt tgctgtggag atcttctccatcttggcatc 2340 tagtgcaggg atgctgggat gcatctttgc acccaaagtt tacatcattttaatgagacc 2400 agacagaaat tcgatccaca aaatcaggga gaaatcatat ttctgaaaaggtatttcagg 2460 aattctgtca aatgtaaagt tgatacatac accccaaata tttagttacagagcatatat 2520 ctagttttag aatcactctc actggttcct ctagttaagc atagaagtaccatatgtact 2580 gatcttgcat atgttgtcta taaaatctta caatcattca tttgcttagtatcttctgga 2640 agaagtaaaa ttttcaaata actagtacaa ttttattcat tattttgctttcatgaggat 2700 ttccccctgg taacttcaaa taaattttat aagtcagttg aatatataaccttacataga 2760 aagtgagttc taggacagac agggattata catagaaaca aactaactaaaaatcaacaa 2820 agatgaaatc agaacacatt ttcttatttc cagtaggaac acatacttgacagaatactg 2880 tctttttttc agctgctctt taagatattg gccaatagtc taagctgaaaatgttcttta 2940 tctactctca aatacaaaaa tattatatcc aacaatggac agaatctgagaactcctgtg 3000 gttgagttag ggaatagttg gaagatactg agaaggaggt gacccataggaatacaaagc 3060 agtctcaact aacctggaca accaaggtcc ctcagacact gagccactaacaagtcagcc 3120 tactccagct gttatgaggc ccccaaaaca tatgcaacat aggattgcctggtccagcct 3180 cagcaagaga atacacacct aaccacagag agacttcccc aagggattggggaggtctgg 3240 ggtttggaga gttgcggatt gtcccttgat gattggaagg aggtattggatgagaatgaa 3300 tcagggggaa gactaggaag gggataatga tggaactgta aaaaaaaaaa3350 14 380 PRT Homo sapiens flap structure-specific endonuclease 1(FEN1) 5′-3′ exonuclease 14 Met Gly Ile Gln Gly Leu Ala Lys Leu Ile AlaAsp Val Ala Pro Ser 1 5 10 15 Ala Ile Arg Glu Asn Asp Ile Lys Ser TyrPhe Gly Arg Lys Val Ala 20 25 30 Ile Asp Ala Ser Met Ser Ile Tyr Gln PheLeu Ile Ala Val Arg Gln 35 40 45 Gly Gly Asp Val Leu Gln Asn Glu Glu GlyGlu Thr Thr Ser His Leu 50 55 60 Met Gly Met Phe Tyr Arg Thr Ile Arg MetMet Glu Asn Gly Ile Lys 65 70 75 80 Pro Val Tyr Val Phe Asp Gly Lys ProPro Gln Leu Lys Ser Gly Glu 85 90 95 Leu Ala Lys Arg Ser Glu Arg Arg AlaGlu Ala Glu Lys Gln Leu Gln 100 105 110 Gln Ala Gln Ala Ala Gly Ala GluGln Glu Val Glu Lys Phe Thr Lys 115 120 125 Arg Leu Val Lys Val Thr LysGln His Asn Asp Glu Cys Lys His Leu 130 135 140 Leu Ser Leu Met Gly IlePro Tyr Leu Asp Ala Pro Ser Glu Ala Glu 145 150 155 160 Ala Ser Cys AlaAla Leu Val Lys Ala Gly Lys Val Tyr Ala Ala Ala 165 170 175 Thr Glu AspMet Asp Cys Leu Thr Phe Gly Ser Pro Val Leu Met Arg 180 185 190 His LeuThr Ala Ser Glu Ala Lys Lys Leu Pro Ile Gln Glu Phe His 195 200 205 LeuSer Arg Ile Leu Gln Glu Leu Gly Leu Asn Gln Glu Gln Phe Val 210 215 220Asp Leu Cys Ile Leu Leu Gly Ser Asp Tyr Cys Glu Ser Ile Arg Gly 225 230235 240 Ile Gly Pro Lys Arg Ala Val Asp Leu Ile Gln Lys His Lys Ser Ile245 250 255 Glu Glu Ile Val Arg Arg Leu Asp Pro Asn Lys Tyr Pro Val ProGlu 260 265 270 Asn Trp Leu His Lys Glu Ala His Gln Leu Phe Leu Glu ProGlu Val 275 280 285 Leu Asp Pro Glu Ser Val Glu Leu Lys Trp Ser Glu ProAsn Glu Glu 290 295 300 Glu Leu Ile Lys Phe Met Cys Gly Glu Lys Gln PheSer Glu Glu Arg 305 310 315 320 Ile Arg Ser Gly Val Lys Arg Leu Ser LysSer Arg Gln Gly Ser Thr 325 330 335 Gln Gly Arg Leu Asp Asp Phe Phe LysVal Thr Gly Ser Leu Ser Ser 340 345 350 Ala Lys Arg Lys Glu Pro Glu ProLys Gly Ser Thr Lys Lys Lys Ala 355 360 365 Lys Thr Gly Ala Ala Gly LysPhe Lys Arg Gly Lys 370 375 380 15 4276 DNA Homo sapiens REV1 dCMPtransferase 15 agagccaccg cggagcgcgc gcggggttgg ttgccgcgag cgtgggggagcgtggaccgc 60 ggcgctgctc agcggtgggg ctgccttccc ccggccctcc tccctggtccctggcgaggg 120 cactggcggc ggcggggccg gggtccgcaa ggccggagaa ggccgccgggcccgggcatg 180 gtggtctggg gcaacgcgga agaagctcca ccatgaggcg aggtggatggaggaagcgag 240 ctgaaaatga tggctgggaa acatggggtg ggtatatggc tgccaaggtccagaaattgg 300 aggaacagtt tcgatcagat gctgctatgc agaaggatgg gacttcatctacaattttta 360 gtggagttgc catctatgtt aatggataca cagatccttc cgctgaggaattgagaaaac 420 taatgatgtt gcatggaggt caataccatg tatattattc cagatctaaaacaacacata 480 ttattgccac aaatcttccc aatgccaaaa ttaaagaatt aaagggggaaaaagtaattc 540 gaccagaatg gattgtggaa agcatcaaag ctggacgact cctctcctacattccatatc 600 agctgtacac caagcagtcc agtgtgcaga aaggtctcag ctttaatcctgtatgcagac 660 ctgaggatcc tctgccaggt ccaagcaata tagccaaaca gctcaacaacagggtaaatc 720 acatcgttaa gaagattgaa acggaaaatg aagtcaaagt caatggcatgaacagttgga 780 atgaagaaga tgaaaataat gattttagtt ttgtggatct ggagcagacctctccgggaa 840 ggaaacagaa tggaattccg catcccagag ggagcactgc catttttaatggacacactc 900 ctagctctaa tggtgcctta aagacacagg attgcttggt gcccatggtcaacagtgttg 960 ccagcaggct ttctccagcc ttttcccagg aggaggataa ggctgagaagagcagcactg 1020 atttcagaga ctgcactctg cagcagttgc agcaaagcac cagaaacacagatgctttgc 1080 ggaatccaca cagaactaat tctttctcat tatcaccttt gcacagtaacactaaaatca 1140 atggtgctca ccactccact gttcaggggc cttcaagcac aaaaagcacttcttcagtat 1200 ctacgtttag caaggcagca ccttcagtgc catccaaacc ttcagactgcaattttattt 1260 caaacttcta ttctcattca agactgcatc acatatcaat gtggaagtgtgaattgactg 1320 agtttgtcaa taccctacaa agacaaagta atggtatctt tccaggaagggaaaagttaa 1380 aaaaaatgaa aacaggcagg tctgcacttg ttgtaactga cacaggagatatgtcagtat 1440 tgaattctcc cagacatcag agctgtataa tgcatgttga tatggattgcttctttgtat 1500 cagtgggtat acgaaataga ccagatctca aaggaaaacc agtggctgttacaagtaaca 1560 gaggcacagg aagggcacct ttacgtcctg gcgctaaccc ccagctggagtggcagtatt 1620 accagaataa aatcctgaaa ggcaaagcag cagatatacc agattcatcattgtgggaga 1680 atccagattc tgcgcaagca aatggaattg attctgtttt gtcaagggctgaaattgcat 1740 cttgtagtta tgaggccagg caacttggca ttaagaacgg aatgttttttgggcatgcta 1800 aacaactatg tcctaatctt caagctgttc catacgattt tcatgcatataaggaagtcg 1860 cacaaacatt gtatgaaaca ttggcaagct acactcataa cattgaagctgtcagttgtg 1920 atgaagcgct ggtagacatt accgaaatcc ttgcagagac caaacttactcctgatgaat 1980 ttgcaaatgc tgttcgtatg gaaatcaaag accagacgaa atgtgctgcctctgttggaa 2040 ttggttctaa tattctcctg gctagaatgg caactagaaa agcaaaaccagatgggcagt 2100 accacctaaa accagaagaa gtagatgatt ttatcagagg ccagctagtgaccaatctac 2160 caggagttgg acattcaatg gaatctaagt tggcatcttt gggaattaaaacttgtggag 2220 acttgcagta tatgaccatg gcaaaactcc aaaaagaatt tggtcccaaaacaggtcaga 2280 tgctttatag gttctgccgt ggcttggatg atagaccagt tcgaactgaaaaggaaagaa 2340 aatctgtttc agctgagatc aactatggaa taaggtttac tcagccaaaagaggcagaag 2400 cttttcttct gagtctttca gaagaaattc aaagaagact agaagccactggcatgaagg 2460 gtaaacgtct aactctcaaa atcatggtac gaaagcctgg ggctcctgtagaaactgcaa 2520 aatttggagg ccatggaatt tgtgataaca ttgccaggac tgtaactcttgaccaggcaa 2580 cagataatgc aaaaataatt ggaaaggcga tgctaaacat gtttcatacaatgaaactaa 2640 atatatcaga tatgagaggg gttgggattc acgtgaatca gttggttccaactaatctga 2700 acccttccac atgtcccagt cgcccatcag ttcagtcaag ccactttcctagtgggtcat 2760 actctgtccg tgatgtcttc caagttcaga aagctaagaa atccaccgaagaggagcaca 2820 aagaagtatt tcgggctgct gtggatctgg aaatatcatc tgcttctagaacttgcactt 2880 tcttgccacc ttttcctgca catctgccga ccagtcctga tactaacaaggctgagtctt 2940 cagggaaatg gaatggtcta catactcctg tcagtgtgca gtcgagacttaacctgagta 3000 tagaggtccc gtcaccttcc cagctggatc agtctgtttt agaagcacttccacctgatc 3060 tccgggaaca agtagagcaa gtctgtgctg tccagcaagc agagtcacatggcgacaaaa 3120 agaaagaacc agtaaatggc tgtaatacag gaattttgcc acaaccagttgggacagtct 3180 tgttgcaaat accagaacct caagaatcga acagtgacgc aggaataaatttaatagccc 3240 ttccagcatt ttcacaggtg gaccctgagg tatttgctgc ccttcctgctgaacttcaga 3300 gggagctgaa agcagcgtat gatcaaagac aaaggcaggg cgagaacagcactcaccagc 3360 agtcagccag cgcatctgtg ccaaagaatc ctttacttca tctaaaggcagcagtgaaag 3420 aaaagaaaag aaacaagaag aaaaaaacca ttggttcacc aaaaaggattcagagtcctt 3480 tgaataacaa gctgcttaac agtcctgcaa aaactctgcc aggggcctgtggcagtcccc 3540 agaagttaat tgatgggttt ctaaaacatg aaggacctcc tgcagagaaacccctggaag 3600 aactctctgc ttctacttca ggtgtgccag gcctttctag tttgcagtctgacccagctg 3660 gctgtgtgag acctccagca cccaatctag ctggagctgt tgaattcaatgatgtgaaga 3720 ccttgctcag agaatggata actacaattt cagatccaat ggaagaagacattctccaag 3780 ttgtgaaata ctgtactgat ctaatagaag aaaaagattt ggaaaaactggatctagtta 3840 taaaatacat gaaaaggctg atgcagcaat cggtggaatc ggtttggaatatggcatttg 3900 actttattct tgacaatgtc caggtggttt tacaacaaac ttatggaagcacattaaaag 3960 ttacataaat attaccagag agcctgatgc tctctgatag ctgtgccataagtgcttgtg 4020 aggtatttgc aaagtgcatg atagtaatgc tcggagtttt tataattttaaatttctttt 4080 aaagcaagtg ttttgtacat ttcttttcaa aaagtgccaa atttgtcagtattgcatgta 4140 aataattgtg ttaattattt tactgtagca tagattctat ttacaaaatgtttgtttata 4200 aagttttatg gatttttaca gtgaagtgtt tacagttgtt taataaagaactgtatgtaa 4260 aaaaaaaaaa aaaaaa 4276 16 1251 PRT Homo sapiens REV1dCMP transferase 16 Met Arg Arg Gly Gly Trp Arg Lys Arg Ala Glu Asn AspGly Trp Glu 1 5 10 15 Thr Trp Gly Gly Tyr Met Ala Ala Lys Val Gln LysLeu Glu Glu Gln 20 25 30 Phe Arg Ser Asp Ala Ala Met Gln Lys Asp Gly ThrSer Ser Thr Ile 35 40 45 Phe Ser Gly Val Ala Ile Tyr Val Asn Gly Tyr ThrAsp Pro Ser Ala 50 55 60 Glu Glu Leu Arg Lys Leu Met Met Leu His Gly GlyGln Tyr His Val 65 70 75 80 Tyr Tyr Ser Arg Ser Lys Thr Thr His Ile IleAla Thr Asn Leu Pro 85 90 95 Asn Ala Lys Ile Lys Glu Leu Lys Gly Glu LysVal Ile Arg Pro Glu 100 105 110 Trp Ile Val Glu Ser Ile Lys Ala Gly ArgLeu Leu Ser Tyr Ile Pro 115 120 125 Tyr Gln Leu Tyr Thr Lys Gln Ser SerVal Gln Lys Gly Leu Ser Phe 130 135 140 Asn Pro Val Cys Arg Pro Glu AspPro Leu Pro Gly Pro Ser Asn Ile 145 150 155 160 Ala Lys Gln Leu Asn AsnArg Val Asn His Ile Val Lys Lys Ile Glu 165 170 175 Thr Glu Asn Glu ValLys Val Asn Gly Met Asn Ser Trp Asn Glu Glu 180 185 190 Asp Glu Asn AsnAsp Phe Ser Phe Val Asp Leu Glu Gln Thr Ser Pro 195 200 205 Gly Arg LysGln Asn Gly Ile Pro His Pro Arg Gly Ser Thr Ala Ile 210 215 220 Phe AsnGly His Thr Pro Ser Ser Asn Gly Ala Leu Lys Thr Gln Asp 225 230 235 240Cys Leu Val Pro Met Val Asn Ser Val Ala Ser Arg Leu Ser Pro Ala 245 250255 Phe Ser Gln Glu Glu Asp Lys Ala Glu Lys Ser Ser Thr Asp Phe Arg 260265 270 Asp Cys Thr Leu Gln Gln Leu Gln Gln Ser Thr Arg Asn Thr Asp Ala275 280 285 Leu Arg Asn Pro His Arg Thr Asn Ser Phe Ser Leu Ser Pro LeuHis 290 295 300 Ser Asn Thr Lys Ile Asn Gly Ala His His Ser Thr Val GlnGly Pro 305 310 315 320 Ser Ser Thr Lys Ser Thr Ser Ser Val Ser Thr PheSer Lys Ala Ala 325 330 335 Pro Ser Val Pro Ser Lys Pro Ser Asp Cys AsnPhe Ile Ser Asn Phe 340 345 350 Tyr Ser His Ser Arg Leu His His Ile SerMet Trp Lys Cys Glu Leu 355 360 365 Thr Glu Phe Val Asn Thr Leu Gln ArgGln Ser Asn Gly Ile Phe Pro 370 375 380 Gly Arg Glu Lys Leu Lys Lys MetLys Thr Gly Arg Ser Ala Leu Val 385 390 395 400 Val Thr Asp Thr Gly AspMet Ser Val Leu Asn Ser Pro Arg His Gln 405 410 415 Ser Cys Ile Met HisVal Asp Met Asp Cys Phe Phe Val Ser Val Gly 420 425 430 Ile Arg Asn ArgPro Asp Leu Lys Gly Lys Pro Val Ala Val Thr Ser 435 440 445 Asn Arg GlyThr Gly Arg Ala Pro Leu Arg Pro Gly Ala Asn Pro Gln 450 455 460 Leu GluTrp Gln Tyr Tyr Gln Asn Lys Ile Leu Lys Gly Lys Ala Ala 465 470 475 480Asp Ile Pro Asp Ser Ser Leu Trp Glu Asn Pro Asp Ser Ala Gln Ala 485 490495 Asn Gly Ile Asp Ser Val Leu Ser Arg Ala Glu Ile Ala Ser Cys Ser 500505 510 Tyr Glu Ala Arg Gln Leu Gly Ile Lys Asn Gly Met Phe Phe Gly His515 520 525 Ala Lys Gln Leu Cys Pro Asn Leu Gln Ala Val Pro Tyr Asp PheHis 530 535 540 Ala Tyr Lys Glu Val Ala Gln Thr Leu Tyr Glu Thr Leu AlaSer Tyr 545 550 555 560 Thr His Asn Ile Glu Ala Val Ser Cys Asp Glu AlaLeu Val Asp Ile 565 570 575 Thr Glu Ile Leu Ala Glu Thr Lys Leu Thr ProAsp Glu Phe Ala Asn 580 585 590 Ala Val Arg Met Glu Ile Lys Asp Gln ThrLys Cys Ala Ala Ser Val 595 600 605 Gly Ile Gly Ser Asn Ile Leu Leu AlaArg Met Ala Thr Arg Lys Ala 610 615 620 Lys Pro Asp Gly Gln Tyr His LeuLys Pro Glu Glu Val Asp Asp Phe 625 630 635 640 Ile Arg Gly Gln Leu ValThr Asn Leu Pro Gly Val Gly His Ser Met 645 650 655 Glu Ser Lys Leu AlaSer Leu Gly Ile Lys Thr Cys Gly Asp Leu Gln 660 665 670 Tyr Met Thr MetAla Lys Leu Gln Lys Glu Phe Gly Pro Lys Thr Gly 675 680 685 Gln Met LeuTyr Arg Phe Cys Arg Gly Leu Asp Asp Arg Pro Val Arg 690 695 700 Thr GluLys Glu Arg Lys Ser Val Ser Ala Glu Ile Asn Tyr Gly Ile 705 710 715 720Arg Phe Thr Gln Pro Lys Glu Ala Glu Ala Phe Leu Leu Ser Leu Ser 725 730735 Glu Glu Ile Gln Arg Arg Leu Glu Ala Thr Gly Met Lys Gly Lys Arg 740745 750 Leu Thr Leu Lys Ile Met Val Arg Lys Pro Gly Ala Pro Val Glu Thr755 760 765 Ala Lys Phe Gly Gly His Gly Ile Cys Asp Asn Ile Ala Arg ThrVal 770 775 780 Thr Leu Asp Gln Ala Thr Asp Asn Ala Lys Ile Ile Gly LysAla Met 785 790 795 800 Leu Asn Met Phe His Thr Met Lys Leu Asn Ile SerAsp Met Arg Gly 805 810 815 Val Gly Ile His Val Asn Gln Leu Val Pro ThrAsn Leu Asn Pro Ser 820 825 830 Thr Cys Pro Ser Arg Pro Ser Val Gln SerSer His Phe Pro Ser Gly 835 840 845 Ser Tyr Ser Val Arg Asp Val Phe GlnVal Gln Lys Ala Lys Lys Ser 850 855 860 Thr Glu Glu Glu His Lys Glu ValPhe Arg Ala Ala Val Asp Leu Glu 865 870 875 880 Ile Ser Ser Ala Ser ArgThr Cys Thr Phe Leu Pro Pro Phe Pro Ala 885 890 895 His Leu Pro Thr SerPro Asp Thr Asn Lys Ala Glu Ser Ser Gly Lys 900 905 910 Trp Asn Gly LeuHis Thr Pro Val Ser Val Gln Ser Arg Leu Asn Leu 915 920 925 Ser Ile GluVal Pro Ser Pro Ser Gln Leu Asp Gln Ser Val Leu Glu 930 935 940 Ala LeuPro Pro Asp Leu Arg Glu Gln Val Glu Gln Val Cys Ala Val 945 950 955 960Gln Gln Ala Glu Ser His Gly Asp Lys Lys Lys Glu Pro Val Asn Gly 965 970975 Cys Asn Thr Gly Ile Leu Pro Gln Pro Val Gly Thr Val Leu Leu Gln 980985 990 Ile Pro Glu Pro Gln Glu Ser Asn Ser Asp Ala Gly Ile Asn Leu Ile995 1000 1005 Ala Leu Pro Ala Phe Ser Gln Val Asp Pro Glu Val Phe AlaAla Leu 1010 1015 1020 Pro Ala Glu Leu Gln Arg Glu Leu Lys Ala Ala TyrAsp Gln Arg Gln 1025 1030 1035 1040 Arg Gln Gly Glu Asn Ser Thr His GlnGln Ser Ala Ser Ala Ser Val 1045 1050 1055 Pro Lys Asn Pro Leu Leu HisLeu Lys Ala Ala Val Lys Glu Lys Lys 1060 1065 1070 Arg Asn Lys Lys LysLys Thr Ile Gly Ser Pro Lys Arg Ile Gln Ser 1075 1080 1085 Pro Leu AsnAsn Lys Leu Leu Asn Ser Pro Ala Lys Thr Leu Pro Gly 1090 1095 1100 AlaCys Gly Ser Pro Gln Lys Leu Ile Asp Gly Phe Leu Lys His Glu 1105 11101115 1120 Gly Pro Pro Ala Glu Lys Pro Leu Glu Glu Leu Ser Ala Ser ThrSer 1125 1130 1135 Gly Val Pro Gly Leu Ser Ser Leu Gln Ser Asp Pro AlaGly Cys Val 1140 1145 1150 Arg Pro Pro Ala Pro Asn Leu Ala Gly Ala ValGlu Phe Asn Asp Val 1155 1160 1165 Lys Thr Leu Leu Arg Glu Trp Ile ThrThr Ile Ser Asp Pro Met Glu 1170 1175 1180 Glu Asp Ile Leu Gln Val ValLys Tyr Cys Thr Asp Leu Ile Glu Glu 1185 1190 1195 1200 Lys Asp Leu GluLys Leu Asp Leu Val Ile Lys Tyr Met Lys Arg Leu 1205 1210 1215 Met GlnGln Ser Val Glu Ser Val Trp Asn Met Ala Phe Asp Phe Ile 1220 1225 1230Leu Asp Asn Val Gln Val Val Leu Gln Gln Thr Tyr Gly Ser Thr Leu 12351240 1245 Lys Val Thr 1250 17 2957 DNA Homo sapiens apyrimidinicendonuclease 1 (APE1), AP endonuclease 1, HAP1 17 ctgcagatag cactgggaaagacaccgcgg aactcccgcg agcgagaccc gccaaggccc 60 ctccagggac ctgtcttcctaacgtccagg gagcccgagc caactcgcgc cttacattcg 120 tatccgtttt cctatctctttcccgtggtc agcccagcct tctccactgt ttttttcctc 180 ttgcacagag ttagaatcttaagtcagtgt cacacaatgt gctgtgcatc tggcacaacg 240 ataaacagcc gagggagggttggggactaa gtgcctagag aattagagga gggaggcgag 300 gctaagcgtc cgtcacgtggtgtcagacag accaatcacg cgcattcttc ggccacgaca 360 agcgcgcctc tgatcacgtgaccaggtccg ctacccacgt gggggctcag cgtgcaccct 420 tctttgtgct cgggttaggaggagctaggc tgccatcggg ccggtgcaga tacggggttg 480 ctcttttgct cataagaggggcttcgctgg cagtctgaac ggcaagcttg agtcaggacc 540 cttaattaag atcctcaattggctggaggg cagatctcgc gagtagggta caaggcacta 600 tgaaatgatc tagtttcgtgggtgaggggc tgaagggcct atgatgcacg gaggcgggga 660 aaggatttag agataacgtggtttaaaggc gggacctggt gcggggacgc tccttgggag 720 gagtcttctc ccagccttagctggtttcat gatttctttg cgtctgtagg caacgcggta 780 aaaatattgc ttcggtgggtgacgcggtac agctgcccaa gggcgttcgt aacgggaatg 840 ccgaagcgtg ggaaaaagggagcggtggcg gaagacgggg atgagctcag gacaggtaag 900 ggaatgaaat cagcccttcttcctagaagc tgcggcgggg gtgtttgtca ttcccttgat 960 gtacggtaag tacgggccgactcatttttg caggggtttg tgaagaagtc gcaggaaccg 1020 taggctttcg ttgggtctatagttaacgcc ggatcgcagt tggaaaccac cagctttttg 1080 tcagtatata ttactcattttatagagcca gaggccaaga agagtaagac ggccgcaaag 1140 aaaaatgaca aagaggcagcaggagagggc ccagccctgt atgaggaccc cccagatcag 1200 aaaacctcac ccagtggcaaacctgccaca ctcaagatct gctcttggaa tgtggatggg 1260 cttcgagcct ggattaagaagaaaggatta gatgtgagtg gaatttgagg gaaagagaca 1320 ttttttagta ttgaatggtcttagggttta gtcacccctt ttctccgttt agccttcagg 1380 ctgttttatt tttctcctgcccgtagtttt ctgtggggct tccccagtct tgccagttgt 1440 atttcctaaa tgtctgttccttcacttcca ttgccatttt cttttttagt gttctctcct 1500 cttcccagaa tgttgcaaaaacctcttcac tatacttcct ccattttatc ttcctgcatt 1560 gcattccata tgaagcatgtcctccattcc attaaccata gcttaaaatc ttagcttgct 1620 atccactgcc tatagaaaaaacacatctcc ttggcatagc atgtaagact ttcttacctc 1680 tctatatttg ttttcatttatctagcttag aattgtttga atattgtgct gcttgactcg 1740 aactccttag gccaagagactgtttaaccc gtgcgtatct atgacttagc atatagatta 1800 ttcaataaat gttctgctgaattgataata cgttttccac ctttcttttc acttacagtg 1860 ggtaaaggaa gaagccccagatatactgtg ccttcaagag accaaatgtt cagagaacaa 1920 actaccagct gaacttcaggagctgcctgg actctctcat caatactggt cagctccttc 1980 ggacaaggaa gggtacagtggcgtgggcct gctttcccgc cagtgcccac tcaaagtttc 2040 ttacggcata ggtgagaccctattgatgcc taatgcctga actcttcaaa accaattgct 2100 aattctctat ctctgccccacctcttgatt gctttccctt ttcttatagt tttttatgct 2160 aattctgttt catttctataggcgatgagg agcatgatca ggaaggccgg gtgattgtgg 2220 ctgaatttga ctcgtttgtgctggtaacag catatgtacc taatgcaggc cgaggtctgg 2280 tacgactgga gtaccggcagcgctgggatg aagcctttcg caagttcctg aagggcctgg 2340 cttcccgaaa gccccttgtgctgtgtggag acctcaatgt ggcacatgaa gaaattgacc 2400 ttcgcaaccc caaggggaacaaaaagaatg ctggcttcac gccacaagag cgccaaggct 2460 tcggggaatt actgcaggctgtgccactgg ctgacagctt taggcacctc taccccaaca 2520 caccctatgc ctacaccttttggacttata tgatgaatgc tcgatccaag aatgttggtt 2580 ggcgccttga ttactttttgttgtcccact ctctgttacc tgcattgtgt gacagcaaga 2640 tccgttccaa ggccctcggcagtgatcact gtcctatcac cctataccta gcactgtgac 2700 accaccccta aatcactttgagcctgggaa ataagccccc tcaactacca ttccttcttt 2760 aaacactctt cagagaaatctgcattctat ttctcatgta taaaactagg aatcctccaa 2820 ccaggctcct gtgatagagttcttttaagc ccaagatttt ttatttgagg gttttttgtt 2880 ttttaaaaaa aaattgaacaaagactacta atgactttgt ttgaattatc cacatgaaaa 2940 taaagagcca tagtttc 295718 318 PRT Homo sapiens apyrimidinic endonuclease 1 (APE1), APendonuclease 1, HAP1 18 Met Pro Lys Arg Gly Lys Lys Gly Ala Val Ala GluAsp Gly Asp Glu 1 5 10 15 Leu Arg Thr Glu Pro Glu Ala Lys Lys Ser LysThr Ala Ala Lys Lys 20 25 30 Asn Asp Lys Glu Ala Ala Gly Glu Gly Pro AlaLeu Tyr Glu Asp Pro 35 40 45 Pro Asp Gln Lys Thr Ser Pro Ser Gly Lys ProAla Thr Leu Lys Ile 50 55 60 Cys Ser Trp Asn Val Asp Gly Leu Arg Ala TrpIle Lys Lys Lys Gly 65 70 75 80 Leu Asp Trp Val Lys Glu Glu Ala Pro AspIle Leu Cys Leu Gln Glu 85 90 95 Thr Lys Cys Ser Glu Asn Lys Leu Pro AlaGlu Leu Gln Glu Leu Pro 100 105 110 Gly Leu Ser His Gln Tyr Trp Ser AlaPro Ser Asp Lys Glu Gly Tyr 115 120 125 Ser Gly Val Gly Leu Leu Ser ArgGln Cys Pro Leu Lys Val Ser Tyr 130 135 140 Gly Ile Gly Asp Glu Glu HisAsp Gln Glu Gly Arg Val Ile Val Ala 145 150 155 160 Glu Phe Asp Ser PheVal Leu Val Thr Ala Tyr Val Pro Asn Ala Gly 165 170 175 Arg Gly Leu ValArg Leu Glu Tyr Arg Gln Arg Trp Asp Glu Ala Phe 180 185 190 Arg Lys PheLeu Lys Gly Leu Ala Ser Arg Lys Pro Leu Val Leu Cys 195 200 205 Gly AspLeu Asn Val Ala His Glu Glu Ile Asp Leu Arg Asn Pro Lys 210 215 220 GlyAsn Lys Lys Asn Ala Gly Phe Thr Pro Gln Glu Ala Gln Gly Phe 225 230 235240 Gly Glu Leu Leu Gln Ala Val Pro Leu Ala Asp Ser Phe Arg His Leu 245250 255 Tyr Pro Asn Thr Pro Tyr Ala Tyr Thr Phe Trp Thr Tyr Met Met Asn260 265 270 Ala Arg Ser Lys Asn Val Gly Trp Arg Leu Asp Tyr Phe Leu LeuSer 275 280 285 His Ser Leu Leu Pro Ala Leu Cys Asp Ser Lys Ile Arg SerLys Ala 290 295 300 Leu Gly Ser Asp His Cys Pro Ile Thr Leu Tyr Leu AlaLeu 305 310 315 19 1161 DNA Homo sapiens cyclin-dependent kinase 3(CDK3), cyclin- dependent protein kinase 19 ccacatggaa gctggaggagcaaccgggag cgctgggctg gggtgcaaat tgcccagtgc 60 cttctgtttc ccaggcagctctgtggccat ggatatgttc cagaaggtag agaagatcgg 120 agagggcacc tatggggtggtgtacaaggc caagaacagg gagacagggc agctggtggc 180 cctgaagaag atcagactggatttggagat ggagggggtc ccaagcactg ccatcaggga 240 gatctcgctg ctcaaggaactgaagcaccc caacatcgtc cgactgctgg acgtggtgca 300 caacgagagg aagctctatctggtgtttga gttcctcagc caggacctga agaagtacat 360 ggactccacc ccaggctcagagctccccct gcacctcatc aagagctacc tcttccagct 420 gctgcagggg gtgagtttctgccactcaca tcgggtcatc caccgagacc tgaagcccca 480 gaacctgctc atcaatgagttgggtgccat caagctggct gacttcggcc tggctcgcgc 540 cttcggggtg cccctgcgcacctacaccca tgaggtggtg acactgtggt atcgcgcccc 600 cgagattctc ttgggcagcaagttctatac cacagctgtg gatatctgga gcattggttg 660 catctttgca gagatggtgactcgaaaagc cctgtttcct ggtgactctg agattgacca 720 gctctttcgt atctttcgtatgctggggac acccagcgaa gacacatggc ccggggtcac 780 ccagctgcct gactataagggcagcttccc taagtggacc aggaagggac tggaagagat 840 tgtgcccaat ctggagccagagggcaggga cctgctcatg caactcctgc agtatgaccc 900 cagccagcgg atcacagccaagactgccct ggcccacccg tacttctcat cccctgagcc 960 ctccccagct gcccgccagtatgtgctgca gcgattccgc cattgagaat gtcaaggcca 1020 cactcagatc ctttctcgagcagcagctgc tgccccagct gcctcctacc cattgccaag 1080 agaggatgca tctggggagagcaaagcact aaggaattca gcatcagcct gcagagggct 1140 gagtctgggt tagtcctgcc c1161 20 305 PRT Homo sapiens cyclin-dependent kinase 3 (CDK3), cyclin-dependent protein kinase 20 Met Asp Met Phe Gln Lys Val Glu Lys Ile GlyGlu Gly Thr Tyr Gly 1 5 10 15 Val Val Tyr Lys Ala Lys Asn Arg Glu ThrGly Gln Leu Val Ala Leu 20 25 30 Lys Lys Ile Arg Leu Asp Leu Glu Met GluGly Val Pro Ser Thr Ala 35 40 45 Ile Arg Glu Ile Ser Leu Leu Lys Glu LeuLys His Pro Asn Ile Val 50 55 60 Arg Leu Leu Asp Val Val His Asn Glu ArgLys Leu Tyr Leu Val Phe 65 70 75 80 Glu Phe Leu Ser Gln Asp Leu Lys LysTyr Met Asp Ser Thr Pro Gly 85 90 95 Ser Glu Leu Pro Leu His Leu Ile LysSer Tyr Leu Phe Gln Leu Leu 100 105 110 Gln Gly Val Ser Phe Cys His SerHis Arg Val Ile His Arg Asp Leu 115 120 125 Lys Pro Gln Asn Leu Leu IleAsn Glu Leu Gly Ala Ile Lys Leu Ala 130 135 140 Asp Phe Gly Leu Ala ArgAla Phe Gly Val Pro Leu Arg Thr Tyr Thr 145 150 155 160 His Glu Val ValThr Leu Trp Tyr Arg Ala Pro Glu Ile Leu Leu Gly 165 170 175 Ser Lys PheTyr Thr Thr Ala Val Asp Ile Trp Ser Ile Gly Cys Ile 180 185 190 Phe AlaGlu Met Val Thr Arg Lys Ala Leu Phe Pro Gly Asp Ser Glu 195 200 205 IleAsp Gln Leu Phe Arg Ile Phe Arg Met Leu Gly Thr Pro Ser Glu 210 215 220Asp Thr Trp Pro Gly Val Thr Gln Leu Pro Asp Tyr Lys Gly Ser Phe 225 230235 240 Pro Lys Trp Thr Arg Lys Gly Leu Glu Glu Ile Val Pro Asn Leu Glu245 250 255 Pro Glu Gly Arg Asp Leu Leu Met Gln Leu Leu Gln Tyr Asp ProSer 260 265 270 Gln Arg Ile Thr Ala Lys Thr Ala Leu Ala His Pro Tyr PheSer Ser 275 280 285 Pro Glu Pro Ser Pro Ala Ala Arg Gln Tyr Val Leu GlnArg Phe Arg 290 295 300 His 305 21 2297 DNA Homo sapiens PIM1 oncogeneserine threonine kinase 21 gcgccgcatc ctggaggttg ggatgctctt gtccaaaatcaactcgcttg cccacctgcg 60 cgcccgcgcc tgcaacgacc tgcacgccac caagctggcgccgggcaagg agaaggagcc 120 cctggagtcg cagtaccagg tgggcccgct actgggcagcggcggcttcg gctcggtcta 180 ctcaggcatc cgcgtctccg acaacttgcc ggtggccatcaaacacgtgg agaaggaccg 240 gatttccgac tggggagagc tgcctaatgg cactcgagtgcccatggaag tggtcctgct 300 gaagaaggtg agctcgggtt tctccggcgt cattaggctcctggactggt tcgagaggcc 360 cgacagtttc gtcctgatcc tggagaggcc cgagccggtgcaagatctct tcgacttcat 420 cacggaaagg ggagccctgc aagaggagct ggcccgcagcttcttctggc aggtgctgga 480 ggccgtgcgg cactgccaca actgcggggt gctccaccgcgacatcaagg acgaaaacat 540 ccttatcgac ctcaatcgcg gcgagctcaa gctcatcgacttcgggtcgg gggcgctgct 600 caaggacacc gtctacacgg acttcgatgg gacccgagtgtatagccctc cagagtggat 660 ccgctaccat cgctaccatg gcaggtcggc ggcagtctggtccctgggga tcctgctgta 720 tgatatggtg tgtggagata ttcctttcga gcatgacgaagagatcatca ggggccaggt 780 tttcttcagg cagagggtct cttcagaatg tcagcatctcattagatggt gcttggccct 840 gagaccatca gataggccaa ccttcgaaga aatccagaaccatccatgga tgcaagatgt 900 tctcctgccc caggaaactg ctgagatcca cctccacagcctgtcgccgg ggcccagcaa 960 atagcagcct ttctggcagg tcctcccctc tcttgtcagatgcccaggag ggaagcttct 1020 gtctccagct ttcccgagta ccagtgacac gtctcgccaagcaggacagt gcttgataca 1080 ggaacaacat ttacaactca ttccagatcc caggcccctggaggctgcct cccaacagtg 1140 gggaagagtg actctccagg ggtcctaggc ctcaactcctcccatagata ctctcttctt 1200 ctcataggtg tccagcattg ctggactctg aaatatcccgggggtggggg gtgggggtgg 1260 gtcagaaccc tgccatggaa ctgtttcctt catcatgagttctgctgaat gccgcgatgg 1320 gtcaggtagg ggggaaacag gttgggatgg gataggactagcaccatttt aagtccctgt 1380 cacctcttcc gactctttct gagtgccttc tgtggggactccggctgtgc tgggagaaat 1440 acttgaactt gcctctttta cctgctgctt ctccaaaaatctgcctgggt tttgttccct 1500 atttttctct cctgtcctcc ctcaccccct ccttcatatgaaaggtgcca tggaagaggc 1560 tacagggcca aacgctgagc cacctgccct tttttctcctcctttagtaa aactccgagt 1620 gaactggtct tcctttttgg tttttactta actgtttcaaagccaagacc tcacacacac 1680 aaaaaatgca caaacaatgc aatcaacaga aaagctgtaaatgtgtgtac agttggcatg 1740 gtagtataca aaaagattgt agtggatcta atttttaagaaattttgcct ttaagttatt 1800 ttacctgttt ttgtttcttg ttttgaaaga tgcgcattctaacctggagg tcaatgttat 1860 gtatttattt atttatttat ttggttccct tcctannnnnnnnnnngctg ctgccctagt 1920 tttctttcct cctttcctcc tctgacttgg ggaccttttgggggagggct gcgacgcttg 1980 ctctgtttgt ggggtgacgg gactcaggcg ggacagtgctgcagctccct ggcttctgtg 2040 gggcccctca cctacttacc caggtgggtc ccggctctgtgggtgatggg gaggggcatt 2100 gctgactgtg tatataggat aattatgaaa agcagttctggatggtgtgc cttccagatc 2160 ctctctgggg ctgtgttttg agcagcaggt agcctgctggttttatctga gtgaaatact 2220 gtacagggga ataaaagaga tcttattttt ttttttatacttggcgtttt ttgaataaaa 2280 accttttgtc ttaaaac 2297 22 313 PRT Homosapiens PIM1 oncogene serine threonine kinase 22 Met Leu Leu Ser Lys IleAsn Ser Leu Ala His Leu Arg Ala Arg Ala 1 5 10 15 Cys Asn Asp Leu HisAla Thr Lys Leu Ala Pro Gly Lys Glu Lys Glu 20 25 30 Pro Leu Glu Ser GlnTyr Gln Val Gly Pro Leu Leu Gly Ser Gly Gly 35 40 45 Phe Gly Ser Val TyrSer Gly Ile Arg Val Ser Asp Asn Leu Pro Val 50 55 60 Ala Ile Lys His ValGlu Lys Asp Arg Ile Ser Asp Trp Gly Glu Leu 65 70 75 80 Pro Asn Gly ThrArg Val Pro Met Glu Val Val Leu Leu Lys Lys Val 85 90 95 Ser Ser Gly PheSer Gly Val Ile Arg Leu Leu Asp Trp Phe Glu Arg 100 105 110 Pro Asp SerPhe Val Leu Ile Leu Glu Arg Pro Glu Pro Val Gln Asp 115 120 125 Leu PheAsp Phe Ile Thr Glu Arg Gly Ala Leu Gln Glu Glu Leu Ala 130 135 140 ArgSer Phe Phe Trp Gln Val Leu Glu Ala Val Arg His Cys His Asn 145 150 155160 Cys Gly Val Leu His Arg Asp Ile Lys Asp Glu Asn Ile Leu Ile Asp 165170 175 Leu Asn Arg Gly Glu Leu Lys Leu Ile Asp Phe Gly Ser Gly Ala Leu180 185 190 Leu Lys Asp Thr Val Tyr Thr Asp Phe Asp Gly Thr Arg Val TyrSer 195 200 205 Pro Pro Glu Trp Ile Arg Tyr His Arg Tyr His Gly Arg SerAla Ala 210 215 220 Val Trp Ser Leu Gly Ile Leu Leu Tyr Asp Met Val CysGly Asp Ile 225 230 235 240 Pro Phe Glu His Asp Glu Glu Ile Ile Arg GlyGln Val Phe Phe Arg 245 250 255 Gln Arg Val Ser Ser Glu Cys Gln His LeuIle Arg Trp Cys Leu Ala 260 265 270 Leu Arg Pro Ser Asp Arg Pro Thr PheGlu Glu Ile Gln Asn His Pro 275 280 285 Trp Met Gln Asp Val Leu Leu ProGln Glu Thr Ala Glu Ile His Leu 290 295 300 His Ser Leu Ser Pro Gly ProSer Lys 305 310 23 3178 DNA Homo sapiens CDC7 cell division cycle 7(CDC7), CDC7 cell division cycle 7-like 1 (CDC7L1) protein serinethreonine kinase 23 gatctcttgg agacggcgac ccaggcatct ggggagccacagaagtcgta ctcccttaaa 60 ccctgctttg ctccccctgt ggatgtaacc ccttagctggcattttgcat ctcaattggc 120 ttgtgatgga ggcgtctttg gggattcaga tggatgagccaatggctttt tctccccagc 180 gtgaccggtt tcaggctgaa ggctctttaa aaaaaaacgagcagaatttt aaacttgcag 240 gtgttaaaaa agatattgag aagctttatg aagctgtaccacagcttagt aatgtgttta 300 agattgagga caaaattgga gaaggcactt tcagctctgtttatttggcc acagcacagt 360 tacaagtagg acctgaagag aaaattgctc taaaacacttgattccaaca agtcatccta 420 taagaattgc agctgaactt cagtgcctaa cagtggctggggggcaagat aatgtcatgg 480 gagttaaata ctgctttagg aagaatgatc atgtagttattgctatgcca tatctggagc 540 atgagtcgtt tttggacatt ctgaattctc tttcctttcaagaagtacgg gaatatatgc 600 ttaatctgtt caaagctttg aaacgcattc atcagtttggtattgttcac cgtgatgtta 660 agcccagcaa ttttttatat aataggcgcc tgaaaaagtatgccttggta gactttggtt 720 tggcccaagg aacccatgat acgaaaatag agcttcttaaatttgtccag tctgaagctc 780 agcaggaaag gtgttcacaa aacaaatccc acataatcacaggaaacaag attccactga 840 gtggcccagt acctaaggag ctggatcagc agtccaccacaaaagcttct gttaaaagac 900 cctacacaaa tgcacaaatt cagattaaac aaggaaaagacggaaaggag ggatctgtag 960 gcctttctgt ccagcgctct gtttttggag aaagaaatttcaatatacac agctccattt 1020 cacatgagag ccctgcagtg aaactcatga agcagtcaaagactgtggat gtactgtcta 1080 gaaagttagc aacaaaaaag aaggctattt ctacgaaagttatgaatagt gctgtgatga 1140 ggaaaactgc cagttcttgc ccagctagcc tgacctgtgactgctatgca acagataaag 1200 tttgtagtat ttgcctttca aggcgtcagc aggttgcccctagggcaggt acaccaggat 1260 tcagagcacc agaggtcttg acaaagtgcc ccaatcaaactacagcaatt gacatgtggt 1320 ctgcaggtgt catatttctt tctttgctta gtggacgatatccattttat aaagcaagtg 1380 atgatttaac tgctttggcc caaattatga caattaggggatccagagaa actatccaag 1440 ctgctaaaac ttttgggaaa tcaatattat gtagcaaagaagttccagca caagacttga 1500 gaaaactctg tgagagactc aggggtatgg attctagcactcccaagtta acaagtgata 1560 tacaagggca tgcttctcat caaccagcta tttcagagaagactgaccat aaagcttctt 1620 gcctcgttca aacacctcca ggacaatact cagggaattcatttaaaaag ggggatagta 1680 atagctgtga gcattgtttt gatgagtata ataccaatttagaaggctgg aatgaggtac 1740 ctgatgaagc ttatgacctg cttgataaac ttctagatctaaatccagct tcaagaataa 1800 cagcagaaga agctttgttg catccatttt ttaaagatatgagcttgtga taatggatct 1860 tcatttaatg tttactgtta tgaggtagaa taaaaaagaatactttgtaa tagccacaag 1920 ttcttgttta gagaccagag caggattaat aatttattttaacattttag tgtttggtgg 1980 cacattctaa aatatagatt aagaatactt aaaatgcctgggatagttct tgggactaac 2040 aacatgatct tctttgagtt aaacctacct aagtagattttaggtgggtt cctattaggt 2100 cagattttta gcttccctaa ttacctttca ctgacatacagaaaaaggag cagttttagt 2160 tttaattaat taaaattaac agatgtgatg aggattaaatgaatcaaaag acttaatttg 2220 tagattcttt tagagttatg agctaggtat agtttggggaaactcaacct ggtgctggtg 2280 ctcttaacaa ttttgtaaat aaagaagata atttccttttctagaggtac atattaggcc 2340 ttttatgaac actaaaacaa tgaggaaatg ttggtcatggggcaaagtat cacttaaaat 2400 tgaattcatc catttttaaa aaacacttca tgaaagcattctggtgtgaa ttgccatttt 2460 tttcttactg gcttctcaat tttcttcctt ctctgcccctacctaaaaca ttctcctcgg 2520 aaattacatg gtgctgacca caaagtttct ggatgttttattaaatattg tacgtgttta 2580 cagttgggaa tttaaaataa tacatacact ggttgataaagggaagctgc aggaccaagg 2640 tgaagattga tagtccaaat gcttttcttt tttgagttgtatattttttc acaccatctt 2700 agatataatt aggtagctgc tgaaaggaaa agtgaatacagaattgacgg tattattgga 2760 gatttttcct ctgcgtagag ccatccagat ctctgtatcctgttttgact aagtcttagg 2820 tgggttggga agacagataa tgaagtaggc aaagagaaaaggacccaaga tagaggttta 2880 tattcagaaa tggtatatat caatgacagc atatcaaacttcctatggga aaaagtctgg 2940 tgggtggtca gctgacagat ttcccattta gtagtcatagaatacagaaa tagtttaggg 3000 acatgtattc attttgttat tttgagcatt gataggtcagtatatctacc taatctgttt 3060 ggtaagtata ggatatataa accattacca ttgatctgtcttatgccata atcttaaaaa 3120 aaaattgaat gctcttgaat ttgtatattc aataaagttatccttttata aaaaaaaa 3178 24 574 PRT Homo sapiens CDC7 cell divisioncycle 7 (CDC7), CDC7 cell division cycle 7-like 1 (CDC7L1) proteinserine threonine kinase 24 Met Glu Ala Ser Leu Gly Ile Gln Met Asp GluPro Met Ala Phe Ser 1 5 10 15 Pro Gln Arg Asp Arg Phe Gln Ala Glu GlySer Leu Lys Lys Asn Glu 20 25 30 Gln Asn Phe Lys Leu Ala Gly Val Lys LysAsp Ile Glu Lys Leu Tyr 35 40 45 Glu Ala Val Pro Gln Leu Ser Asn Val PheLys Ile Glu Asp Lys Ile 50 55 60 Gly Glu Gly Thr Phe Ser Ser Val Tyr LeuAla Thr Ala Gln Leu Gln 65 70 75 80 Val Gly Pro Glu Glu Lys Ile Ala LeuLys His Leu Ile Pro Thr Ser 85 90 95 His Pro Ile Arg Ile Ala Ala Glu LeuGln Cys Leu Thr Val Ala Gly 100 105 110 Gly Gln Asp Asn Val Met Gly ValLys Tyr Cys Phe Arg Lys Asn Asp 115 120 125 His Val Val Ile Ala Met ProTyr Leu Glu His Glu Ser Phe Leu Asp 130 135 140 Ile Leu Asn Ser Leu SerPhe Gln Glu Val Arg Glu Tyr Met Leu Asn 145 150 155 160 Leu Phe Lys AlaLeu Lys Arg Ile His Gln Phe Gly Ile Val His Arg 165 170 175 Asp Val LysPro Ser Asn Phe Leu Tyr Asn Arg Arg Leu Lys Lys Tyr 180 185 190 Ala LeuVal Asp Phe Gly Leu Ala Gln Gly Thr His Asp Thr Lys Ile 195 200 205 GluLeu Leu Lys Phe Val Gln Ser Glu Ala Gln Gln Glu Arg Cys Ser 210 215 220Gln Asn Lys Ser His Ile Ile Thr Gly Asn Lys Ile Pro Leu Ser Gly 225 230235 240 Pro Val Pro Lys Glu Leu Asp Gln Gln Ser Thr Thr Lys Ala Ser Val245 250 255 Lys Arg Pro Tyr Thr Asn Ala Gln Ile Gln Ile Lys Gln Gly LysAsp 260 265 270 Gly Lys Glu Gly Ser Val Gly Leu Ser Val Gln Arg Ser ValPhe Gly 275 280 285 Glu Arg Asn Phe Asn Ile His Ser Ser Ile Ser His GluSer Pro Ala 290 295 300 Val Lys Leu Met Lys Gln Ser Lys Thr Val Asp ValLeu Ser Arg Lys 305 310 315 320 Leu Ala Thr Lys Lys Lys Ala Ile Ser ThrLys Val Met Asn Ser Ala 325 330 335 Val Met Arg Lys Thr Ala Ser Ser CysPro Ala Ser Leu Thr Cys Asp 340 345 350 Cys Tyr Ala Thr Asp Lys Val CysSer Ile Cys Leu Ser Arg Arg Gln 355 360 365 Gln Val Ala Pro Arg Ala GlyThr Pro Gly Phe Arg Ala Pro Glu Val 370 375 380 Leu Thr Lys Cys Pro AsnGln Thr Thr Ala Ile Asp Met Trp Ser Ala 385 390 395 400 Gly Val Ile PheLeu Ser Leu Leu Ser Gly Arg Tyr Pro Phe Tyr Lys 405 410 415 Ala Ser AspAsp Leu Thr Ala Leu Ala Gln Ile Met Thr Ile Arg Gly 420 425 430 Ser ArgGlu Thr Ile Gln Ala Ala Lys Thr Phe Gly Lys Ser Ile Leu 435 440 445 CysSer Lys Glu Val Pro Ala Gln Asp Leu Arg Lys Leu Cys Glu Arg 450 455 460Leu Arg Gly Met Asp Ser Ser Thr Pro Lys Leu Thr Ser Asp Ile Gln 465 470475 480 Gly His Ala Ser His Gln Pro Ala Ile Ser Glu Lys Thr Asp His Lys485 490 495 Ala Ser Cys Leu Val Gln Thr Pro Pro Gly Gln Tyr Ser Gly AsnSer 500 505 510 Phe Lys Lys Gly Asp Ser Asn Ser Cys Glu His Cys Phe AspGlu Tyr 515 520 525 Asn Thr Asn Leu Glu Gly Trp Asn Glu Val Pro Asp GluAla Tyr Asp 530 535 540 Leu Leu Asp Lys Leu Leu Asp Leu Asn Pro Ala SerArg Ile Thr Ala 545 550 555 560 Glu Glu Ala Leu Leu His Pro Phe Phe LysAsp Met Ser Leu 565 570 25 1427 DNA Homo sapiens cyclin-dependent kinase7 (CDK7), kinase subunit of Cdk-activating kinase (CAK), kinasecomponent of transcription factor complex TFIIH 25 tgggtgttgg aggctttaaggtagctttaa attcgtgttg tcctgggagc tcgccctttt 60 cggctggagt cgggctttacggcgccggat ggctctggac gtgaagtctc gggcaaagcg 120 ttatgagaag ctggacttccttggggaggg acagtttgcc accgtttaca aggccagaga 180 taagaatacc aaccaaattgtcgccattaa gaaaatcaaa cttggacata gatcagaagc 240 taaagatggt ataaatagaaccgccttaag agagataaaa ttattacagg agctaagtca 300 tccaaatata attggtctccttgatgcttt tggacataaa tctaatatta gccttgtctt 360 tgattttatg gaaactgatctagaggttat aataaaggat aatagtcttg tgctgacacc 420 atcacacatc aaagcctacatgttgatgac tcttcaagga ttagaatatt tacatcaaca 480 ttggatccta catagggatctgaaaccaaa caacttgttg ctagatgaaa atggagttct 540 aaaactggca gattttggcctggccaaatc ttttgggagc cccaatagag cttatacaca 600 tcaggttgta accaggtggtatcgggcccc cgagttacta tttggagcta ggatgtatgg 660 tgtaggtgtg gacatgtgggctgttggctg tatattagca gagttacttc taagggttcc 720 ttttttgcca ggagattcagaccttgatca gctaacaaga atatttgaaa ctttgggcac 780 accaactgag gaacagtggccggacatgtg tagtcttcca gattatgtga catttaagag 840 tttccctgga atacctttgcatcacatctt cagtgcagca ggagacgact tactagatct 900 catacaaggc ttattcttatttaatccatg tgctcgaatt acggccacac aggcactgaa 960 aatgaagtat ttcagtaatcggccagggcc aacacctgga tgtcagctgc caagaccaaa 1020 ctgtccagtg gaaaccttaaaggagcaatc aaatccagct ttggcaataa aaaggaaaag 1080 aacagaggcc ttagaacaaggaggattgcc caagaaacta attttttaaa gagaacactg 1140 gacaacattt tactactgagggaaatagcc aaaaaggcaa ataatggaaa aatagtaaac 1200 attaagtaaa tgctgtagaagtgagtttgt aaatattcta cacatgtaaa atatgtaaaa 1260 ctatgggtta tttttattaaatgtatttta aaataaaaat ttaattctgg tttttctgat 1320 tagagtccca aagtgagaaaagttcaatac tcttgaaatg tagaattgaa aatgcattag 1380 ggaaaactta ataaaaattattaccagtta tttggaaaaa aaaaaaa 1427 26 346 PRT Homo sapienscyclin-dependent kinase 7 (CDK7), kinase subunit of Cdk-activatingkinase (CAK), kinase component of transcription factor complex TFIIH 26Met Ala Leu Asp Val Lys Ser Arg Ala Lys Arg Tyr Glu Lys Leu Asp 1 5 1015 Phe Leu Gly Glu Gly Gln Phe Ala Thr Val Tyr Lys Ala Arg Asp Lys 20 2530 Asn Thr Asn Gln Ile Val Ala Ile Lys Lys Ile Lys Leu Gly His Arg 35 4045 Ser Glu Ala Lys Asp Gly Ile Asn Arg Thr Ala Leu Arg Glu Ile Lys 50 5560 Leu Leu Gln Glu Leu Ser His Pro Asn Ile Ile Gly Leu Leu Asp Ala 65 7075 80 Phe Gly His Lys Ser Asn Ile Ser Leu Val Phe Asp Phe Met Glu Thr 8590 95 Asp Leu Glu Val Ile Ile Lys Asp Asn Ser Leu Val Leu Thr Pro Ser100 105 110 His Ile Lys Ala Tyr Met Leu Met Thr Leu Gln Gly Leu Glu TyrLeu 115 120 125 His Gln His Trp Ile Leu His Arg Asp Leu Lys Pro Asn AsnLeu Leu 130 135 140 Leu Asp Glu Asn Gly Val Leu Lys Leu Ala Asp Phe GlyLeu Ala Lys 145 150 155 160 Ser Phe Gly Ser Pro Asn Arg Ala Tyr Thr HisGln Val Val Thr Arg 165 170 175 Trp Tyr Arg Ala Pro Glu Leu Leu Phe GlyAla Arg Met Tyr Gly Val 180 185 190 Gly Val Asp Met Trp Ala Val Gly CysIle Leu Ala Glu Leu Leu Leu 195 200 205 Arg Val Pro Phe Leu Pro Gly AspSer Asp Leu Asp Gln Leu Thr Arg 210 215 220 Ile Phe Glu Thr Leu Gly ThrPro Thr Glu Glu Gln Trp Pro Asp Met 225 230 235 240 Cys Ser Leu Pro AspTyr Val Thr Phe Lys Ser Phe Pro Gly Ile Pro 245 250 255 Leu His His IlePhe Ser Ala Ala Gly Asp Asp Leu Leu Asp Leu Ile 260 265 270 Gln Gly LeuPhe Leu Phe Asn Pro Cys Ala Arg Ile Thr Ala Thr Gln 275 280 285 Ala LeuLys Met Lys Tyr Phe Ser Asn Arg Pro Gly Pro Thr Pro Gly 290 295 300 CysGln Leu Pro Arg Pro Asn Cys Pro Val Glu Thr Leu Lys Glu Gln 305 310 315320 Ser Asn Pro Ala Leu Ala Ile Lys Arg Lys Arg Thr Glu Ala Leu Glu 325330 335 Gln Gly Gly Leu Pro Lys Lys Leu Ile Phe 340 345 27 2169 DNA Homosapiens cytokine-inducible kinase (CNK) serine threonine kinase,proliferation-related kinase (PRK), polo-like kinase 3 (PLK3) 27ccgcctccga gtgccttgcg cggacctgag ctggagatgc tggccgggct accgacgtca 60gaccccgggc gcctcatcac ggacccgcgc agcggccgca cctacctcaa aggccgcttg 120ttgggcaagg ggggcttcgc ccgctgctac gaggccactg acacagagac tggcagcgcc 180tacgctgtca aagtcatccc gcagagccgc gtcgccaagc cgcatcagcg cgagaagatc 240ctaaatgaga ttgagctgca ccgagacctg cagcaccgcc acatcgtgcg tttttcgcac 300cactttgagg acgctgacaa catctacatt ttcttggagc tctgcagccg aaagtccctg 360gcccacatct ggaaggcccg gcacaccctg ttggagccag aagtgcgcta ctacctgcgg 420cagatccttt ctggcctcaa gtacttgcac cagcgcggca tcttgcaccg ggacctcaag 480ttgggaaatt ttttcatcac tgagaacatg gaactgaagg tgggggattt tgggctggca 540gcccggttgg agcctccgga gcagaggaag aagaccatct gtggcacccc caactatgtg 600gctccagaag tgctgctgag acagggccac ggccctgaag cggatgtatg gtcactgggc 660tgtgtcatgt acacgctgct ctgcgggagc cctccctttg agacggctga cctgaaggag 720acgtaccgct gcatcaagca ggttcactac acgctgcctg ccagcctctc actgcctgcc 780cggcagctcc tggccgccat ccttcgggcc tcaccccgag accgcccctc tattgaccag 840atcctgcgcc atgacttctt taccaagggc tacacccccg atcgactccc tatcagcagc 900tgcgtgacag tcccagacct gacacccccc aacccagcta ggagtctgtt tgccaaagtt 960accaagagcc tctttggcag aaagaagaag agtaagaatc atgcccagga gagggatgag 1020gtctccggtt tggtgagcgg cctcatgcgc acatccgttg gccatcagga tgccaggcca 1080gaggctccag cagcttctgg cccagcccct gtcagcctgg tagagacagc acctgaagac 1140agctcacccc gtgggacact ggcaagcagt ggagatggat ttgaagaagg tctgactgtg 1200gccacagtag tggagtcagc cctttgtgct ctgagaaatt gtatagcttt catgccccca 1260gcggaacaga acccggcccc cctggcccag ccagagcctc tggtgtgggt cagcaagtgg 1320gttgactact ccaataagtt cggctttggg tatcaactgt ccagccgccg tgtggctgtg 1380ctcttcaacg atggcacaca tatggccctg tcggccaaca gaaagactgt gcactacaat 1440cccaccagca caaagcactt ctccttctcc gtgggtgctg tgccccgggc cctgcagcct 1500cagctgggta tcctgcggta cttcgcctcc tacatggagc agcacctcat gaagggtgga 1560gatctgccca gtgtggaaga ggtagaggta cctgctccgc ccttgctgct gcagtgggtc 1620aagacggatc aggctctcct catgctgttt agtgatggca ctgtccaggt gaacttctac 1680ggggaccaca ccaagctgat tctcagtggc tgggagcccc tccttgtgac ttttgtggcc 1740cgaaatcgta gtgcttgtac ttacctcgct tcccaccttc ggcagctggg ctgctctcca 1800gacctgcggc agcgactccg ctatgctctg cgcctgctcc gggaccgcag cccagcttag 1860gacccaagcc ctgaaggcct gaggcctgtg cctgtcaggc tctggccctt gcctttgtgg 1920ccttccccct tcctttggtg cctcactggg ggctttgggc cgaatccccc agggaatcag 1980ggaccagctt tactggagtt gggggcggct tgtcttcgct ggctcctacc ccatctccaa 2040gataagcctg agccttagct cccagctagg gggcgttatt tatggaccac ttttatttat 2100tgtcagacac ttatttattg ggatgtgagc cccagggggc ctcctcctag gataataaac 2160aattttgca 2169 28 607 PRT Homo sapiens cytokine-inducible kinase (CNK)serine threonine kinase, proliferation-related kinase (PRK), polo-likekinase 3 (PLK3) 28 Met Leu Ala Gly Leu Pro Thr Ser Asp Pro Gly Arg LeuIle Thr Asp 1 5 10 15 Pro Arg Ser Gly Arg Thr Tyr Leu Lys Gly Arg LeuLeu Gly Lys Gly 20 25 30 Gly Phe Ala Arg Cys Tyr Glu Ala Thr Asp Thr GluThr Gly Ser Ala 35 40 45 Tyr Ala Val Lys Val Ile Pro Gln Ser Arg Val AlaLys Pro His Gln 50 55 60 Arg Glu Lys Ile Leu Asn Glu Ile Glu Leu His ArgAsp Leu Gln His 65 70 75 80 Arg His Ile Val Arg Phe Ser His His Phe GluAsp Ala Asp Asn Ile 85 90 95 Tyr Ile Phe Leu Glu Leu Cys Ser Arg Lys SerLeu Ala His Ile Trp 100 105 110 Lys Ala Arg His Thr Leu Leu Glu Pro GluVal Arg Tyr Tyr Leu Arg 115 120 125 Gln Ile Leu Ser Gly Leu Lys Tyr LeuHis Gln Arg Gly Ile Leu His 130 135 140 Arg Asp Leu Lys Leu Gly Asn PhePhe Ile Thr Glu Asn Met Glu Leu 145 150 155 160 Lys Val Gly Asp Phe GlyLeu Ala Ala Arg Leu Glu Pro Pro Glu Gln 165 170 175 Arg Lys Lys Thr IleCys Gly Thr Pro Asn Tyr Val Ala Pro Glu Val 180 185 190 Leu Leu Arg GlnGly His Gly Pro Glu Ala Asp Val Trp Ser Leu Gly 195 200 205 Cys Val MetTyr Thr Leu Leu Cys Gly Ser Pro Pro Phe Glu Thr Ala 210 215 220 Asp LeuLys Glu Thr Tyr Arg Cys Ile Lys Gln Val His Tyr Thr Leu 225 230 235 240Pro Ala Ser Leu Ser Leu Pro Ala Arg Gln Leu Leu Ala Ala Ile Leu 245 250255 Arg Ala Ser Pro Arg Asp Arg Pro Ser Ile Asp Gln Ile Leu Arg His 260265 270 Asp Phe Phe Thr Lys Gly Tyr Thr Pro Asp Arg Leu Pro Ile Ser Ser275 280 285 Cys Val Thr Val Pro Asp Leu Thr Pro Pro Asn Pro Ala Arg SerLeu 290 295 300 Phe Ala Lys Val Thr Lys Ser Leu Phe Gly Arg Lys Lys LysSer Lys 305 310 315 320 Asn His Ala Gln Glu Arg Asp Glu Val Ser Gly LeuVal Ser Gly Leu 325 330 335 Met Arg Thr Ser Val Gly His Gln Asp Ala ArgPro Glu Ala Pro Ala 340 345 350 Ala Ser Gly Pro Ala Pro Val Ser Leu ValGlu Thr Ala Pro Glu Asp 355 360 365 Ser Ser Pro Arg Gly Thr Leu Ala SerSer Gly Asp Gly Phe Glu Glu 370 375 380 Gly Leu Thr Val Ala Thr Val ValGlu Ser Ala Leu Cys Ala Leu Arg 385 390 395 400 Asn Cys Ile Ala Phe MetPro Pro Ala Glu Gln Asn Pro Ala Pro Leu 405 410 415 Ala Gln Pro Glu ProLeu Val Trp Val Ser Lys Trp Val Asp Tyr Ser 420 425 430 Asn Lys Phe GlyPhe Gly Tyr Gln Leu Ser Ser Arg Arg Val Ala Val 435 440 445 Leu Phe AsnAsp Gly Thr His Met Ala Leu Ser Ala Asn Arg Lys Thr 450 455 460 Val HisTyr Asn Pro Thr Ser Thr Lys His Phe Ser Phe Ser Val Gly 465 470 475 480Ala Val Pro Arg Ala Leu Gln Pro Gln Leu Gly Ile Leu Arg Tyr Phe 485 490495 Ala Ser Tyr Met Glu Gln His Leu Met Lys Gly Gly Asp Leu Pro Ser 500505 510 Val Glu Glu Val Glu Val Pro Ala Pro Pro Leu Leu Leu Gln Trp Val515 520 525 Lys Thr Asp Gln Ala Leu Leu Met Leu Phe Ser Asp Gly Thr ValGln 530 535 540 Val Asn Phe Tyr Gly Asp His Thr Lys Leu Ile Leu Ser GlyTrp Glu 545 550 555 560 Pro Leu Leu Val Thr Phe Val Ala Arg Asn Arg SerAla Cys Thr Tyr 565 570 575 Leu Ala Ser His Leu Arg Gln Leu Gly Cys SerPro Asp Leu Arg Gln 580 585 590 Arg Leu Arg Tyr Ala Leu Arg Leu Leu ArgAsp Arg Ser Pro Ala 595 600 605 29 1321 DNA Homo sapiens potentiallyprenylated protein tyrosine phosphatase (PRL-3), protein tyrosinephosphatase type IVA, member 3, isoform 2, transcript variant 2 (PTP4A3)29 tgactatcca gctctgagag acgggagttt ggagttgccc gctttacttt ggttgggttg 60gggggggcgg cgggctgttt tgttcctttt cttttttaag agttgggttt tcttttttaa 120ttatccaaac agtgggcagc ttcctccccc acacccaagt atttgcacaa tatttgtgcg 180gggtatgggg gtgggttttt aaatctcgtt tctcttggac aagcacaggg atctcgttct 240cctcattttt tgggggtgtg tggggacttc tcaggtcgtg tccccagcct tctctgcagt 300cccttctgcc ctgccgggcc cgtcgggagg cgccatggct cggatgaacc gcccggcccc 360ggtggaggtg agctacaaac acatgcgctt cctcatcacc cacaacccca ccaacgccac 420gctcagcacc ttcattgagg acctgaagaa gtacggggct accactgtgg tgcgtgtgtg 480tgaagtgacc tatgacaaaa cgccgctgga gaaggatggc atcaccgttg tggactggcc 540gtttgacgat ggggcgcccc cgcccggcaa ggtagtggaa gactggctga gcctggtgaa 600ggccaagttc tgtgaggccc ccggcagctg cgtggctgtg cactgcgtgg cgggcctggg 660ccggaagcgc cgcggagcca tcaacagcaa gcagctcacc tacctggaga aataccggcc 720caaacagagg ctgcggttca aagacccaca cacgcacaag acccggtgct gcgttatgta 780gctcaggacc ttggctgggc ctggtcgtca tgtaggtcag gaccttggct ggacctggag 840gccctgccca gccctgctct gcccagccca gcaggggctc caggccttgg ctggccccac 900atcgcctttt cctccccgac acctccgtgc acttgtgtcc gaggagcgag gagcccctcg 960ggccctgggt ggcctctggg ccctttctcc tgtctccgcc actccctctg gcggcgctgg 1020ccgtggctct gtctctctga ggtgggtcgg gcgccctctg cccgccccct cccacaccag 1080ccaggctggt ctcctctagc ctgtttgttg tggggtgggg gtatattttg taaccactgg 1140gcccccagcc cctcttttgc gaccccttgt cctgacctgt tctcggcacc ttaaattatt 1200agaccccggg gcagtcaggt gctccggaca cccgaaggca ataaaacagg agccgtgaaa 1260aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1320 a1321 30 148 PRT Homo sapiens potentially prenylated protein tyrosinephosphatase (PRL-3), protein tyrosine phosphatase type IVA, member 3,isoform 2, transcript variant 2 (PTP4A3) 30 Met Ala Arg Met Asn Arg ProAla Pro Val Glu Val Ser Tyr Lys His 1 5 10 15 Met Arg Phe Leu Ile ThrHis Asn Pro Thr Asn Ala Thr Leu Ser Thr 20 25 30 Phe Ile Glu Asp Leu LysLys Tyr Gly Ala Thr Thr Val Val Arg Val 35 40 45 Cys Glu Val Thr Tyr AspLys Thr Pro Leu Glu Lys Asp Gly Ile Thr 50 55 60 Val Val Asp Trp Pro PheAsp Asp Gly Ala Pro Pro Pro Gly Lys Val 65 70 75 80 Val Glu Asp Trp LeuSer Leu Val Lys Ala Lys Phe Cys Glu Ala Pro 85 90 95 Gly Ser Cys Val AlaVal His Cys Val Ala Gly Leu Gly Arg Lys Arg 100 105 110 Arg Gly Ala IleAsn Ser Lys Gln Leu Thr Tyr Leu Glu Lys Tyr Arg 115 120 125 Pro Lys GlnArg Leu Arg Phe Lys Asp Pro His Thr His Lys Thr Arg 130 135 140 Cys CysVal Met 145 31 3696 DNA Homo sapiens serine threonine kinase 2 (STK2,NEK4) 31 ggatcgctat ggcagcggcg tcgtcgcggg ccgggcccca gcaatcccgcccgggcccgg 60 ctgcctcaac agccgccccc actgccccct ctcgggcatg aaccgagcttcttgttgccg 120 cccgctgccc tacccgccgc tgccgccgca tcccgactct gggccagcgctgggaacatg 180 cccctggccg cctactgcta cctgcgggtc gtgggcaagg ggagctatggagaggtgacg 240 cttgtgaagc accggcggga cggcaagcag tatgtcatca aaaaactgaacctccgaaat 300 gcctctagcc gagagcggcg agctgctgaa caggaagccc agctcttgtctcagttgaag 360 catcccaaca ttgtcaccta caaggagtca tgggaaggag gagatggtctgctctacatt 420 gtcatgggct tctgtgaagg aggtgatttg taccgaaagc tcaaggagcagaaagggcag 480 cttctgcctg agaatcaggt ggtagagtgg tttgtacaga tcgccatggctttgcagtat 540 ttacatgaaa aacacatcct tcatcgagat ctgaaaactc aaaatgtcttcctaacaaga 600 acaaacatca tcaaagtagg ggacctagga attgcccgag tgttagagaaccactgtgac 660 atggctagca ccctcattgg cacaccctac tacatgagcc ctgaattgttctcaaacaaa 720 ccctacaact ataagtctga tgtttgggct ctaggatgct gtgtctatgaaatggccacc 780 ttgaagcatg ctttcaatgc aaaagatatg aattctttag tttatcggattattgaagga 840 aagctgccac caatgccaag agattacagc ccagagctgg cagaactgataagaacaatg 900 ctgagcaaaa ggcctgaaga aaggccgtct gtgaggagca tcctgaggcagccttatata 960 aagcggcaaa tctccttctt tttggaggcc acaaagataa aaacctccaaaaataacatt 1020 aaaaatggtg actctcaatc caagcctttt gctacagtgg tttctggagaggcagaatca 1080 aatcatgaag taatccaccc ccaaccactc tcttctgagg gctcccagacatatataatg 1140 ggtgaaggca aatgtttgtc ccaggagaaa cccagggcct ctggtctcttgaagtcacct 1200 gccagtctga aagcccatac ctgcaaacag gacttgagca ataccacagaactagccaca 1260 atcagtagcg taaatattga catcttacct gcaaaaggga gggattcagtgagtgatggc 1320 tttgttcagg agaatcagcc aagatatttg gatgcctcta atgagttaggaggtatatgc 1380 agtatttctc aagtggaaga ggagatgctg caggacaaca ctaaatccagtgcccagcct 1440 gaaaacctga ttcccatgtg gtcctctgac attgtcactg gggaaaagaatgaaccagtg 1500 aagcctctgc agcccctaat caaagaacaa aagccaaagg accagagtcttgccctgtcg 1560 cccaagctgg agtgcagtgg cacaatcttg gctcacagca acctccgcctcctgggttca 1620 agtgattctc cagcctcagc ctcccgagta gctgggatta caggcgtgtgccaccacgcc 1680 caggatcaag ttgctggtga atgtattata gaaaaacagg gcagaatccacccagattta 1740 cagccacaca actctgggtc tgaaccttcc ctgtctcgac agcgacggcaaaagaggaga 1800 gaacagactg agcacagagg ggaaaagaga caggtccgca gagatctctttgctttccaa 1860 gagtcgcctc ctcgattttt gccttctcat cccattgttg ggaaagtggatgtcacatca 1920 acacaaaaag aggctgaaaa ccaacgtaga gtggtcactg ggtctgtgagcagttcaagg 1980 agcagtgaga tgtcatcatc aaaggatcga ccattatcag ccagagagaggaggcgacta 2040 aagcagtcac aggaagaaat gtcctcttca ggcccttcag tgaggaaagcgtctctgagt 2100 gtagcagggc caggaaaacc ccaggaagaa gaccagccct tgcctgcccgacggctctcc 2160 tctgactgca gcgtcactca ggaaaggaaa cagattcatt gtctgtctgaggatgagtta 2220 agttcttcta caagttcaac tgataagtca gatggggatt acggggaagggaaaggtcag 2280 acaaatgaaa ttaatgcctt ggtacaattg atgactcaga ccctgaaactggattctaaa 2340 gagagctgtg aagatgtccc ggtagcaaac ccagtgtcag aattcaaacttcatcggaaa 2400 tatcgggaca cactgatact tcatgggaag gttgcagaag aggcagaggaaatccatttt 2460 aaagagctac cttcagctat tatgccaggt tctgaaaaga tcaggagactagttgaagtc 2520 ttgagaactg atgtaattcg tggcctggga gttcagcttt tagagcaggtgtatgatctt 2580 ttggaggagg aggatgaatt tgatagagag gtacgtttgc gggagcacatgggtgaaaag 2640 tatacaactt acagtgtgaa agctcgccag ttgaaatttt ttgaagaaaacatgaatttt 2700 tgagcatttg tcctaatctg ctgccagaat taaagaccta tttttagaggattttggctt 2760 aaaaagcaag ggcaaacagt catttggaag ccactcacca ctgttttatatctctttttt 2820 atatctcttt ggcgtttccc tacagaaaag aaattggaca gaacagaataatatgaagca 2880 ggatcacaaa agaaaaaaaa ctttggcttt catattctct ttgtgaggacaaatctgttg 2940 tttgtttgat tactgtttac tgagccttaa tccaccaagt ttatatttagaattttattt 3000 ttttaaggta ctaattaact taaacacaga gctataaaat gctggattgaaaattttata 3060 ttgtaatgta gagataaaag cagtaggaga aacaaatgac ataatatgtcgtcataattc 3120 ctgctattgt taataacctt aaggagtagt tgataaatta taaaattttaaaaagtcaat 3180 tcagttctag aaatagattt aaagaatatg aagttctatc tagtacttgagcagctgtat 3240 ttcttttcta cacattgatg gacttttaat attttattct catttaatataaacctcatc 3300 tagggtatat acaaattaaa actgagacac attggctttg taaatcagtatgtttttaca 3360 taatggtttt gttagattta tttttccatc agtgaaaaca tttcttaagcacaaatttca 3420 tttccattta agcaatttgt aagcaaagtc caggtccatt tagtttttggatatatttaa 3480 tgtttgtctc ctgaagtttg tcttcatgta ctgtaagata ttagttgtctttccatgttt 3540 taaatgtatg attatatagc acatatttta ttagttgttt aataagaggtaatacccatc 3600 taggaaagaa attttatgaa gttaaataca agtcttgaat agtacattttcacttctgta 3660 ttcgagggac tctaaaaata aatattgctc cagaaa 3696 32 841 PRTHomo sapiens serine threonine kinase 2 (STK2, NEK4) 32 Met Pro Leu AlaAla Tyr Cys Tyr Leu Arg Val Val Gly Lys Gly Ser 1 5 10 15 Tyr Gly GluVal Thr Leu Val Lys His Arg Arg Asp Gly Lys Gln Tyr 20 25 30 Val Ile LysLys Leu Asn Leu Arg Asn Ala Ser Ser Arg Glu Arg Arg 35 40 45 Ala Ala GluGln Glu Ala Gln Leu Leu Ser Gln Leu Lys His Pro Asn 50 55 60 Ile Val ThrTyr Lys Glu Ser Trp Glu Gly Gly Asp Gly Leu Leu Tyr 65 70 75 80 Ile ValMet Gly Phe Cys Glu Gly Gly Asp Leu Tyr Arg Lys Leu Lys 85 90 95 Glu GlnLys Gly Gln Leu Leu Pro Glu Asn Gln Val Val Glu Trp Phe 100 105 110 ValGln Ile Ala Met Ala Leu Gln Tyr Leu His Glu Lys His Ile Leu 115 120 125His Arg Asp Leu Lys Thr Gln Asn Val Phe Leu Thr Arg Thr Asn Ile 130 135140 Ile Lys Val Gly Asp Leu Gly Ile Ala Arg Val Leu Glu Asn His Cys 145150 155 160 Asp Met Ala Ser Thr Leu Ile Gly Thr Pro Tyr Tyr Met Ser ProGlu 165 170 175 Leu Phe Ser Asn Lys Pro Tyr Asn Tyr Lys Ser Asp Val TrpAla Leu 180 185 190 Gly Cys Cys Val Tyr Glu Met Ala Thr Leu Lys His AlaPhe Asn Ala 195 200 205 Lys Asp Met Asn Ser Leu Val Tyr Arg Ile Ile GluGly Lys Leu Pro 210 215 220 Pro Met Pro Arg Asp Tyr Ser Pro Glu Leu AlaGlu Leu Ile Arg Thr 225 230 235 240 Met Leu Ser Lys Arg Pro Glu Glu ArgPro Ser Val Arg Ser Ile Leu 245 250 255 Arg Gln Pro Tyr Ile Lys Arg GlnIle Ser Phe Phe Leu Glu Ala Thr 260 265 270 Lys Ile Lys Thr Ser Lys AsnAsn Ile Lys Asn Gly Asp Ser Gln Ser 275 280 285 Lys Pro Phe Ala Thr ValVal Ser Gly Glu Ala Glu Ser Asn His Glu 290 295 300 Val Ile His Pro GlnPro Leu Ser Ser Glu Gly Ser Gln Thr Tyr Ile 305 310 315 320 Met Gly GluGly Lys Cys Leu Ser Gln Glu Lys Pro Arg Ala Ser Gly 325 330 335 Leu LeuLys Ser Pro Ala Ser Leu Lys Ala His Thr Cys Lys Gln Asp 340 345 350 LeuSer Asn Thr Thr Glu Leu Ala Thr Ile Ser Ser Val Asn Ile Asp 355 360 365Ile Leu Pro Ala Lys Gly Arg Asp Ser Val Ser Asp Gly Phe Val Gln 370 375380 Glu Asn Gln Pro Arg Tyr Leu Asp Ala Ser Asn Glu Leu Gly Gly Ile 385390 395 400 Cys Ser Ile Ser Gln Val Glu Glu Glu Met Leu Gln Asp Asn ThrLys 405 410 415 Ser Ser Ala Gln Pro Glu Asn Leu Ile Pro Met Trp Ser SerAsp Ile 420 425 430 Val Thr Gly Glu Lys Asn Glu Pro Val Lys Pro Leu GlnPro Leu Ile 435 440 445 Lys Glu Gln Lys Pro Lys Asp Gln Ser Leu Ala LeuSer Pro Lys Leu 450 455 460 Glu Cys Ser Gly Thr Ile Leu Ala His Ser AsnLeu Arg Leu Leu Gly 465 470 475 480 Ser Ser Asp Ser Pro Ala Ser Ala SerArg Val Ala Gly Ile Thr Gly 485 490 495 Val Cys His His Ala Gln Asp GlnVal Ala Gly Glu Cys Ile Ile Glu 500 505 510 Lys Gln Gly Arg Ile His ProAsp Leu Gln Pro His Asn Ser Gly Ser 515 520 525 Glu Pro Ser Leu Ser ArgGln Arg Arg Gln Lys Arg Arg Glu Gln Thr 530 535 540 Glu His Arg Gly GluLys Arg Gln Val Arg Arg Asp Leu Phe Ala Phe 545 550 555 560 Gln Glu SerPro Pro Arg Phe Leu Pro Ser His Pro Ile Val Gly Lys 565 570 575 Val AspVal Thr Ser Thr Gln Lys Glu Ala Glu Asn Gln Arg Arg Val 580 585 590 ValThr Gly Ser Val Ser Ser Ser Arg Ser Ser Glu Met Ser Ser Ser 595 600 605Lys Asp Arg Pro Leu Ser Ala Arg Glu Arg Arg Arg Leu Lys Gln Ser 610 615620 Gln Glu Glu Met Ser Ser Ser Gly Pro Ser Val Arg Lys Ala Ser Leu 625630 635 640 Ser Val Ala Gly Pro Gly Lys Pro Gln Glu Glu Asp Gln Pro LeuPro 645 650 655 Ala Arg Arg Leu Ser Ser Asp Cys Ser Val Thr Gln Glu ArgLys Gln 660 665 670 Ile His Cys Leu Ser Glu Asp Glu Leu Ser Ser Ser ThrSer Ser Thr 675 680 685 Asp Lys Ser Asp Gly Asp Tyr Gly Glu Gly Lys GlyGln Thr Asn Glu 690 695 700 Ile Asn Ala Leu Val Gln Leu Met Thr Gln ThrLeu Lys Leu Asp Ser 705 710 715 720 Lys Glu Ser Cys Glu Asp Val Pro ValAla Asn Pro Val Ser Glu Phe 725 730 735 Lys Leu His Arg Lys Tyr Arg AspThr Leu Ile Leu His Gly Lys Val 740 745 750 Ala Glu Glu Ala Glu Glu IleHis Phe Lys Glu Leu Pro Ser Ala Ile 755 760 765 Met Pro Gly Ser Glu LysIle Arg Arg Leu Val Glu Val Leu Arg Thr 770 775 780 Asp Val Ile Arg GlyLeu Gly Val Gln Leu Leu Glu Gln Val Tyr Asp 785 790 795 800 Leu Leu GluGlu Glu Asp Glu Phe Asp Arg Glu Val Arg Leu Arg Glu 805 810 815 His MetGly Glu Lys Tyr Thr Thr Tyr Ser Val Lys Ala Arg Gln Leu 820 825 830 LysPhe Phe Glu Glu Asn Met Asn Phe 835 840 33 1513 DNA Homo sapiens serinethreonine protein kinase NKIAMRE, mitogen-activated proteinkinase/cyclin- dependent kinase-related protein kinase NKIATRE homologue33 atggagatgt atgaaaccct tggaaaagtg ggagagggaa gttacggaac agtcatgaaa 60tgtaaacata agaatactgg gcagatagtg gccattaaga tattttatga gagaccagaa 120caatctgtca acaaaattgc gatgagagaa ataaagtttc taaagcaatt tcatcacgaa 180aacctggtca atctgattga agtttttaga cagaaaaaga aaattcattt ggtatttgaa 240tttattgacc acacagtatt agatgagtta caacattatt gtcatggact agagagtaag 300cgacttagaa aatacctctt ccagatcctt cgagcaattg actatcttca cagtaataat 360atcattcatc gagatataaa acctgagaat attttagtat cccagtcagg aattactaag 420ctctgtgatt ttggttttgc acgaacacta gcagctcctg gggacattta tacggactat 480gtggccacac gctggtatag agctcccgaa ttagtattaa aagatacttc ttatggaaaa 540cctgtggata tctgggcttt gggctgtatg atcattgaga tggccactgg aaatccctat 600cttcctagta gttctgattt ggatttactc cataaaattg ttttgaaagt gggcaatttg 660tcacctcact tgcagaatat cttttccaag agccccattt ttgctggggt agttcttcct 720caagttcaac accccaaaaa tgcaagaaaa aaatatccaa agcttaatgg attgttggca 780gatatagttc atgcttgttt acaaattgat cctgctgaca ggatatcatc tagtgatctt 840ttgcatcatg agtattttac tagagatgga tttattgaaa aattcatgcc agaactgaaa 900gctaaattac tgcaggaagc aaaagtcaat tcattaataa agccaaaaga gagttctaaa 960gaaaatgaac tcaggaaaga tgaaagaaaa acagtttata ccaatacact gctaagtagt 1020tcagttttgg gagaggaaat agaaaaagag aaaaagccca aggagatcaa agtcagagtt 1080attaaagtca aaggaggaag aggagatatc tcagaaccaa aaaagaaaga gtatgaaggt 1140ggacttggtc aacaggatgc aaatgaaaat gttcatccta tgtctccaga tacaaaactt 1200gtaaccattg aaccaccaaa ccctatcaat cccagcacta actgtaatgg cttgaaagaa 1260aatccacatt gcggaggttc tgtaacaatg ccacccatca atctaactaa cagtaatttg 1320atggctgcaa atctcagttc aaatctcttt caccccagtg tgaggtgagc tgtaacagag 1380aagaaaccta aataatacaa cattcctgta taatggtatt tcaaagaatc gtgttcatag 1440tgtctgtatg taaactgaac ttgaagaaaa tatattgaaa ttaaagctgt ataatgggcc 1500aaaaaaaaaa aaa 1513 34 455 PRT Homo sapiens serine threonine proteinkinase NKIAMRE, mitogen-activated protein kinase/cyclin- dependentkinase-related protein kinase NKIATRE homologue 34 Met Glu Met Tyr GluThr Leu Gly Lys Val Gly Glu Gly Ser Tyr Gly 1 5 10 15 Thr Val Met LysCys Lys His Lys Asn Thr Gly Gln Ile Val Ala Ile 20 25 30 Lys Ile Phe TyrGlu Arg Pro Glu Gln Ser Val Asn Lys Ile Ala Met 35 40 45 Arg Glu Ile LysPhe Leu Lys Gln Phe His His Glu Asn Leu Val Asn 50 55 60 Leu Ile Glu ValPhe Arg Gln Lys Lys Lys Ile His Leu Val Phe Glu 65 70 75 80 Phe Ile AspHis Thr Val Leu Asp Glu Leu Gln His Tyr Cys His Gly 85 90 95 Leu Glu SerLys Arg Leu Arg Lys Tyr Leu Phe Gln Ile Leu Arg Ala 100 105 110 Ile AspTyr Leu His Ser Asn Asn Ile Ile His Arg Asp Ile Lys Pro 115 120 125 GluAsn Ile Leu Val Ser Gln Ser Gly Ile Thr Lys Leu Cys Asp Phe 130 135 140Gly Phe Ala Arg Thr Leu Ala Ala Pro Gly Asp Ile Tyr Thr Asp Tyr 145 150155 160 Val Ala Thr Arg Trp Tyr Arg Ala Pro Glu Leu Val Leu Lys Asp Thr165 170 175 Ser Tyr Gly Lys Pro Val Asp Ile Trp Ala Leu Gly Cys Met IleIle 180 185 190 Glu Met Ala Thr Gly Asn Pro Tyr Leu Pro Ser Ser Ser AspLeu Asp 195 200 205 Leu Leu His Lys Ile Val Leu Lys Val Gly Asn Leu SerPro His Leu 210 215 220 Gln Asn Ile Phe Ser Lys Ser Pro Ile Phe Ala GlyVal Val Leu Pro 225 230 235 240 Gln Val Gln His Pro Lys Asn Ala Arg LysLys Tyr Pro Lys Leu Asn 245 250 255 Gly Leu Leu Ala Asp Ile Val His AlaCys Leu Gln Ile Asp Pro Ala 260 265 270 Asp Arg Ile Ser Ser Ser Asp LeuLeu His His Glu Tyr Phe Thr Arg 275 280 285 Asp Gly Phe Ile Glu Lys PheMet Pro Glu Leu Lys Ala Lys Leu Leu 290 295 300 Gln Glu Ala Lys Val AsnSer Leu Ile Lys Pro Lys Glu Ser Ser Lys 305 310 315 320 Glu Asn Glu LeuArg Lys Asp Glu Arg Lys Thr Val Tyr Thr Asn Thr 325 330 335 Leu Leu SerSer Ser Val Leu Gly Glu Glu Ile Glu Lys Glu Lys Lys 340 345 350 Pro LysGlu Ile Lys Val Arg Val Ile Lys Val Lys Gly Gly Arg Gly 355 360 365 AspIle Ser Glu Pro Lys Lys Lys Glu Tyr Glu Gly Gly Leu Gly Gln 370 375 380Gln Asp Ala Asn Glu Asn Val His Pro Met Ser Pro Asp Thr Lys Leu 385 390395 400 Val Thr Ile Glu Pro Pro Asn Pro Ile Asn Pro Ser Thr Asn Cys Asn405 410 415 Gly Leu Lys Glu Asn Pro His Cys Gly Gly Ser Val Thr Met ProPro 420 425 430 Ile Asn Leu Thr Asn Ser Asn Leu Met Ala Ala Asn Leu SerSer Asn 435 440 445 Leu Phe His Pro Ser Val Arg 450 455 35 3504 DNA Homosapiens HBO1 histone acetyltransferase, MYST histone acetyltransferase 2(MYST2) 35 gccgctgccc gaatcggaac cgtcgggccg cagccgccgg caatgccgcgaaggaagagg 60 aatgcaggca gtagttcaga tggaaccgaa gattccgatt tttctacagatctcgagcac 120 acagacagtt cagaaagtga tggcacatcc cgacgatctg ctcgagtcacccgctcctca 180 gccaggctaa gccagagttc tcaagattcc agtcctgttc gaaatctgcagtcttttggc 240 actgaggagc ctgcttactc taccagaaga gtgacccgta gtcagcagcagcctacccca 300 gtgacaccga aaaaataccc tcttcggcag actcgttcat ctggttcagaaactgagcaa 360 gtggttgatt tttcagatag agaaactaaa aatacagctg atcatgatgagtcaccgcct 420 cgaactccaa ctggaaatgc gccttcttct gagtctgaca tagatatctccagccccaat 480 gtatctcacg atgagagcat tgccaaggac atgtccctga aggactcaggcagtgatctc 540 tctcatcgcc ccaagcgccg tcgcttccat gaaagctaca acttcaatatgaagtgtcct 600 acaccaggct gtaactctct aggacacctt acaggaaaac atgagagacatttctccatc 660 tcaggatgcc cactgtatca taacctctca gctgacgaat gcaaggtgagagcacagagc 720 cgggataagc agatagaaga aaggatgctg tctcacaggc aagatgacaacaacaggcat 780 gcaaccaggc accaggcacc aacggagagg cagcttcgat ataaggaaaaagtggctgaa 840 ctcaggaaga aaagaaattc tggactgagc aaagaacaga aagagaaatatatggaacac 900 agacagacct atgggaacac acgggaacct cttttagaaa acctgacaagcgagtatgac 960 ttggatcttt tccgaagagc acaagcccgg gcttcagagg atttggagaagttaaggctg 1020 caaggccaaa tcacagaggg aagcaacatg attaaaacaa ttgcttttggccgctatgag 1080 cttgatacct ggtatcattc tccatatcct gaagaatatg cacggctgggacgtctctat 1140 atgtgtgaat tctgtttaaa atatatgaag agccaaacga tactccgccggcacatggcc 1200 aaatgtgtgt ggaaacaccc acctggtgat gagatatatc gcaaaggttcaatctctgtg 1260 tttgaagtgg atggcaagaa aaacaagatc tactgccaaa acctgtgcctgttggccaaa 1320 ctttttctgg accacaagac attatattat gatgtggagc ccttcctgttctatgttatg 1380 acagaggcgg acaacactgg ctgtcacctg attggatatt tttctaaggaaaagaattca 1440 ttcctcaact acaacgtctc ctgtatcctt actatgcctc agtacatgagacagggctat 1500 ggcaagatgc ttattgattt cagttatttg ctttccaaag tcgaagaaaaagttggctcc 1560 ccagaacgtc cactctcaga tctggggctt ataagctatc gcagttactggaaagaagta 1620 cttctccgct acctgcataa ttttcaaggc aaagagattt ctatcaaagaaatcagtcag 1680 gagacggctg tgaatcctgt ggacattgtc agcactctgc aagcccttcagatgctcaaa 1740 tactggaagg gaaaacacct agttttaaag agacaggacc tgattgatgagtggatagcc 1800 aaagaggcca aaaggtccaa ctccaataaa accatggatc ccagctgcttaaaatggacc 1860 cctcccaagg gcacttaaag tgacctgtca ttccgagcca gcgaaccccagcagtaggaa 1920 tccgtaccct agggatctgt ctgtcatttc tctgttgctc ttgtgattggcaagtacagt 1980 atcctttggg aaggccatcc ccctcaggac tgtcctggct ccgacctttgtgtacactgc 2040 agacgctggt tctgaggaac tgttgtttcg gcctcagtga ggttgcctggatgggatctg 2100 tattagactt gagtgcaggt ctctcagcac tgacccaagg agttctgttatggtactgta 2160 cctgtccagt cactggttct ctcctcatgt cctctcgccc catgaggttgtgttgtgtct 2220 tctaagcgtg gtactagtgc ttgccacctg gtcaccagac ctccaaatatggctgccacc 2280 accaggacct ttccagttac tccttatatg tgtgttctat ggaggggcagggaaaaggtg 2340 gcacttgtga gtgtgtgtgg attggcaggg ggtccattca ctttgggttccatcttgctt 2400 taaatttctt cattttgatt aagagacctc tttttgatct gtattgggctaaccagagcc 2460 aaatactttt gaagagtttc ccagggacta gtcatggtaa tagcatataattgatctgaa 2520 tgagatggag agaagaatga aggggtggtg gttctgggtt tgatttgagttcacctgtgg 2580 gcagtgggca gtgggcagtg tcttggtgaa agggaacgga tactactttttgcctcaccg 2640 taaagtactc actagtaaat atttccttct ctctttactc ccactttttacgtttgcagg 2700 tgccaaagta atgtccactt ttccctttca tgctgcatat taactggttaattatactgc 2760 agaaaccttt tcacctccac tagtctgata cagtacatct gtacttccatataccttgca 2820 ctgattttgt ctgagtgccc tgggagaagt agaaaatgat tgaaagtgacttccgtatct 2880 cagcccatga ctcagcaagg cagaatggcc acccctgcca aagtttgcttctcttttcaa 2940 cagtgcctca ccctccctct aggattaaag tgcttctgcc cttccacgaactcctcctcc 3000 atttcctttt tgggatttgt caccatcctt ctattctctg gtcttctatttttggtgttg 3060 ttcaagtgaa ggaagagatg ttccctctaa tttctctcta gcccattataacctgctatc 3120 ttggggcaac ttttgatgta tgacatgtca cccttcccaa cttggtctcctccaacatgc 3180 tgtcttcatg tggagccctc accacaatcc ctgactccgg tcatttgtgcctttctcttg 3240 tcatctctgt acactactta tattcactgt gggttggggg agctaattttaagcatgttc 3300 agtggcagct cccctccagt ttcagtgtca ctgttaaaat ttatcaaaaagcaacttcac 3360 taggggtttt cttaagggat aaaggccttt tacagaagct aaacccttccccacatgtgg 3420 tagaatgtgc tcttctatat ctactcctca ataaagcatg ttctctgctcaaaaaaaaaa 3480 aaaaaaaaaa aaaaaaaaaa aaaa 3504 36 611 PRT Homo sapiensHBO1 histone acetyltransferase, MYST histone acetyltransferase 2 (MYST2)36 Met Pro Arg Arg Lys Arg Asn Ala Gly Ser Ser Ser Asp Gly Thr Glu 1 510 15 Asp Ser Asp Phe Ser Thr Asp Leu Glu His Thr Asp Ser Ser Glu Ser 2025 30 Asp Gly Thr Ser Arg Arg Ser Ala Arg Val Thr Arg Ser Ser Ala Arg 3540 45 Leu Ser Gln Ser Ser Gln Asp Ser Ser Pro Val Arg Asn Leu Gln Ser 5055 60 Phe Gly Thr Glu Glu Pro Ala Tyr Ser Thr Arg Arg Val Thr Arg Ser 6570 75 80 Gln Gln Gln Pro Thr Pro Val Thr Pro Lys Lys Tyr Pro Leu Arg Gln85 90 95 Thr Arg Ser Ser Gly Ser Glu Thr Glu Gln Val Val Asp Phe Ser Asp100 105 110 Arg Glu Thr Lys Asn Thr Ala Asp His Asp Glu Ser Pro Pro ArgThr 115 120 125 Pro Thr Gly Asn Ala Pro Ser Ser Glu Ser Asp Ile Asp IleSer Ser 130 135 140 Pro Asn Val Ser His Asp Glu Ser Ile Ala Lys Asp MetSer Leu Lys 145 150 155 160 Asp Ser Gly Ser Asp Leu Ser His Arg Pro LysArg Arg Arg Phe His 165 170 175 Glu Ser Tyr Asn Phe Asn Met Lys Cys ProThr Pro Gly Cys Asn Ser 180 185 190 Leu Gly His Leu Thr Gly Lys His GluArg His Phe Ser Ile Ser Gly 195 200 205 Cys Pro Leu Tyr His Asn Leu SerAla Asp Glu Cys Lys Val Arg Ala 210 215 220 Gln Ser Arg Asp Lys Gln IleGlu Glu Arg Met Leu Ser His Arg Gln 225 230 235 240 Asp Asp Asn Asn ArgHis Ala Thr Arg His Gln Ala Pro Thr Glu Arg 245 250 255 Gln Leu Arg TyrLys Glu Lys Val Ala Glu Leu Arg Lys Lys Arg Asn 260 265 270 Ser Gly LeuSer Lys Glu Gln Lys Glu Lys Tyr Met Glu His Arg Gln 275 280 285 Thr TyrGly Asn Thr Arg Glu Pro Leu Leu Glu Asn Leu Thr Ser Glu 290 295 300 TyrAsp Leu Asp Leu Phe Arg Arg Ala Gln Ala Arg Ala Ser Glu Asp 305 310 315320 Leu Glu Lys Leu Arg Leu Gln Gly Gln Ile Thr Glu Gly Ser Asn Met 325330 335 Ile Lys Thr Ile Ala Phe Gly Arg Tyr Glu Leu Asp Thr Trp Tyr His340 345 350 Ser Pro Tyr Pro Glu Glu Tyr Ala Arg Leu Gly Arg Leu Tyr MetCys 355 360 365 Glu Phe Cys Leu Lys Tyr Met Lys Ser Gln Thr Ile Leu ArgArg His 370 375 380 Met Ala Lys Cys Val Trp Lys His Pro Pro Gly Asp GluIle Tyr Arg 385 390 395 400 Lys Gly Ser Ile Ser Val Phe Glu Val Asp GlyLys Lys Asn Lys Ile 405 410 415 Tyr Cys Gln Asn Leu Cys Leu Leu Ala LysLeu Phe Leu Asp His Lys 420 425 430 Thr Leu Tyr Tyr Asp Val Glu Pro PheLeu Phe Tyr Val Met Thr Glu 435 440 445 Ala Asp Asn Thr Gly Cys His LeuIle Gly Tyr Phe Ser Lys Glu Lys 450 455 460 Asn Ser Phe Leu Asn Tyr AsnVal Ser Cys Ile Leu Thr Met Pro Gln 465 470 475 480 Tyr Met Arg Gln GlyTyr Gly Lys Met Leu Ile Asp Phe Ser Tyr Leu 485 490 495 Leu Ser Lys ValGlu Glu Lys Val Gly Ser Pro Glu Arg Pro Leu Ser 500 505 510 Asp Leu GlyLeu Ile Ser Tyr Arg Ser Tyr Trp Lys Glu Val Leu Leu 515 520 525 Arg TyrLeu His Asn Phe Gln Gly Lys Glu Ile Ser Ile Lys Glu Ile 530 535 540 SerGln Glu Thr Ala Val Asn Pro Val Asp Ile Val Ser Thr Leu Gln 545 550 555560 Ala Leu Gln Met Leu Lys Tyr Trp Lys Gly Lys His Leu Val Leu Lys 565570 575 Arg Gln Asp Leu Ile Asp Glu Trp Ile Ala Lys Glu Ala Lys Arg Ser580 585 590 Asn Ser Asn Lys Thr Met Asp Pro Ser Cys Leu Lys Trp Thr ProPro 595 600 605 Lys Gly Thr 610 37 21 DNA Artificial SequenceDescription of Artificial SequenceCK2-specific siRNA molecule 37aacattgaat tagatccacg t 21 38 21 DNA Artificial Sequence Description ofArtificial SequencePIM1- specific siRNA molecule 38 aaaactccgagtgaactggt c 21 39 21 DNA Artificial Sequence Description of ArtificialSequenceHBO1- specific siRNA molecule 39 aactgagcaa gtggttgatt t 21 40409 PRT Homo sapiens CDC7 cell division cycle 7 (CDC7), CDC7 celldivision cycle 7-like 1 (CDC7L1) protein serine threonine kinase 40 MetGlu Ala Ser Leu Gly Ile Gln Met Asp Glu Pro Met Ala Phe Ser 1 5 10 15Pro Gln Arg Asp Arg Phe Gln Ala Glu Gly Ser Leu Lys Lys Asn Glu 20 25 30Gln Asn Phe Lys Leu Ala Gly Val Lys Lys Asp Ile Glu Lys Leu Tyr 35 40 45Glu Ala Val Pro Gln Leu Ser Asn Val Phe Lys Ile Glu Asp Lys Ile 50 55 60Gly Glu Gly Thr Phe Ser Ser Val Tyr Leu Ala Thr Ala Gln Leu Gln 65 70 7580 Val Gly Pro Glu Glu Lys Ile Ala Leu Lys His Leu Ile Pro Thr Ser 85 9095 His Pro Ile Arg Ile Ala Ala Glu Leu Gln Cys Leu Thr Val Ala Gly 100105 110 Gly Gln Asp Asn Val Met Gly Val Lys Tyr Cys Phe Arg Lys Asn Asp115 120 125 His Val Val Ile Ala Met Pro Tyr Leu Glu His Glu Ser Phe LeuAsp 130 135 140 Ile Leu Asn Ser Leu Ser Phe Gln Glu Val Arg Glu Tyr MetLeu Asn 145 150 155 160 Leu Phe Lys Ala Leu Lys Arg Ile His Gln Phe GlyIle Val His Arg 165 170 175 Asp Val Lys Pro Ser Asn Phe Leu Tyr Asn ArgArg Leu Lys Lys Tyr 180 185 190 Ala Leu Val Asp Phe Gly Leu Ala Gln GlyThr His Asp Thr Lys Ile 195 200 205 Glu Leu Leu Lys Phe Val Gln Ser GluAla Gln Gln Glu Arg Cys Ser 210 215 220 Gln Asn Lys Ser His Ile Ile ThrGly Asn Lys Ile Pro Leu Ser Gly 225 230 235 240 Pro Val Pro Lys Glu LeuAsp Gln Gln Ser Thr Thr Lys Ala Ser Val 245 250 255 Lys Arg Pro Tyr ThrAsn Ala Gln Ile Gln Ile Lys Gln Gly Lys Asp 260 265 270 Gly Lys Glu GlySer Val Gly Leu Ser Val Gln Arg Ser Val Phe Gly 275 280 285 Glu Arg AsnPhe Asn Ile His Ser Ser Ile Ser His Glu Ser Pro Ala 290 295 300 Val LysLeu Met Lys Gln Ser Lys Thr Val Asp Val Leu Ser Arg Lys 305 310 315 320Leu Ala Thr Lys Lys Lys Ala Ile Ser Thr Lys Val Met Asn Ser Ala 325 330335 Val Met Arg Lys Thr Ala Ser Ser Cys Pro Ala Ser Leu Thr Cys Asp 340345 350 Cys Tyr Ala Thr Asp Lys Val Cys Ser Ile Cys Leu Ser Arg Arg Gln355 360 365 Gln Val Ala Pro Arg Ala Gly Thr Pro Gly Phe Arg Ala Pro GluVal 370 375 380 Leu Thr Lys Cys Pro Asn Gln Thr Thr Ala Ile Asp Met TrpSer Ala 385 390 395 400 Gly Val Ile Phe Leu Ser Leu Leu Ser 405 41 314PRT Saccharomyces cerevisiae CDC7 41 Met Thr Ser Lys Thr Lys Asn Ile AspAsp Ile Pro Pro Glu Ile Lys 1 5 10 15 Glu Glu Met Ile Gln Leu Tyr HisAsp Leu Pro Gly Ile Glu Asn Glu 20 25 30 Tyr Lys Leu Ile Asp Lys Ile GlyGlu Gly Thr Phe Ser Ser Val Tyr 35 40 45 Lys Ala Lys Asp Ile Thr Gly LysIle Thr Lys Lys Phe Ala Ser His 50 55 60 Phe Trp Asn Tyr Gly Ser Asn TyrVal Ala Leu Lys Lys Ile Tyr Val 65 70 75 80 Thr Ser Ser Pro Gln Arg IleTyr Asn Glu Leu Asn Leu Leu Tyr Ile 85 90 95 Met Thr Gly Ser Ser Arg ValAla Pro Leu Cys Asp Ala Lys Arg Val 100 105 110 Arg Asp Gln Val Ile AlaVal Leu Pro Tyr Tyr Pro His Glu Glu Phe 115 120 125 Arg Thr Phe Tyr ArgAsp Leu Pro Ile Lys Gly Ile Lys Lys Tyr Ile 130 135 140 Trp Glu Leu LeuArg Ala Leu Lys Phe Val His Ser Lys Gly Ile Ile 145 150 155 160 His ArgAsp Ile Lys Pro Thr Asn Phe Leu Phe Asn Leu Glu Leu Gly 165 170 175 ArgGly Val Leu Val Asp Phe Gly Leu Ala Glu Ala Gln Met Asp Tyr 180 185 190Lys Ser Met Ile Ser Ser Gln Asn Asp Tyr Asp Asn Tyr Ala Asn Thr 195 200205 Asn His Asp Gly Gly Tyr Ser Met Arg Asn His Glu Gln Phe Cys Pro 210215 220 Cys Ile Met Arg Asn Gln Tyr Ser Pro Asn Ser His Asn Gln Thr Pro225 230 235 240 Pro Met Val Thr Ile Gln Asn Gly Lys Val Val His Leu AsnAsn Val 245 250 255 Asn Gly Val Asp Leu Thr Lys Gly Tyr Pro Lys Asn GluThr Arg Arg 260 265 270 Ile Lys Arg Ala Asn Arg Ala Gly Thr Arg Gly PheArg Ala Pro Glu 275 280 285 Val Leu Met Lys Cys Gly Ala Gln Ser Thr LysIle Asp Ile Trp Ser 290 295 300 Val Gly Val Ile Leu Leu Ser Leu Leu Gly305 310 42 294 PRT Artificial Sequence Description of ArtificialSequenceprotein kinase consensus sequence 42 Tyr Glu Leu Leu Glu Lys LeuGly Glu Gly Ser Phe Gly Lys Val Tyr 1 5 10 15 Lys Ala Lys His Lys AspLys Thr Gly Lys Ile Val Ala Val Lys Ile 20 25 30 Leu Lys Lys Glu Lys GluSer Ile Lys Glu Lys Arg Phe Leu Arg Glu 35 40 45 Ile Gln Ile Leu Lys ArgLeu Ser His Pro Asn Ile Val Arg Leu Ile 50 55 60 Gly Val Phe Glu Asp ThrAsp Asp His Leu Tyr Leu Val Met Glu Tyr 65 70 75 80 Met Glu Gly Gly AspLeu Phe Asp Tyr Leu Arg Arg Asn Gly Gly Pro 85 90 95 Leu Ser Glu Lys GluAla Lys Lys Ile Ala Leu Gln Ile Leu Arg Gly 100 105 110 Leu Glu Tyr LeuHis Ser Asn Gly Ile Val His Arg Asp Leu Lys Pro 115 120 125 Glu Asn IleLeu Leu Asp Glu Asn Asp Gly Thr Val Lys Ile Ala Asp 130 135 140 Phe GlyLeu Ala Arg Leu Leu Glu Ser Ser Ser Lys Leu Thr Thr Phe 145 150 155 160Val Gly Thr Pro Trp Tyr Met Met Ala Pro Glu Val Ile Leu Glu Gly 165 170175 Arg Gly Tyr Ser Ser Lys Val Asp Val Trp Ser Leu Gly Val Ile Leu 180185 190 Tyr Glu Leu Leu Thr Gly Gly Pro Leu Phe Pro Gly Ala Asp Leu Pro195 200 205 Ala Phe Thr Gly Gly Asp Glu Val Asp Gln Leu Ile Ile Phe ValLeu 210 215 220 Lys Leu Pro Phe Ser Asp Glu Leu Pro Lys Thr Arg Ile AspPro Leu 225 230 235 240 Glu Glu Leu Phe Arg Ile Ile Lys Arg Pro Gly LeuArg Leu Pro Leu 245 250 255 Pro Ser Asn Cys Ser Glu Glu Leu Lys Asp LeuLeu Lys Lys Cys Leu 260 265 270 Asn Lys Asp Pro Ser Lys Arg Pro Gly SerAla Thr Ala Lys Glu Ile 275 280 285 Leu Asn His Pro Trp Phe 290 43 253PRT Homo sapiens cytokine-inducible kinase (CNK) serine threoninekinase, proliferation-related kinase (PRK), polo-like kinase 3 (PLK3) 43Tyr Leu Lys Gly Arg Leu Leu Gly Lys Gly Gly Phe Ala Arg Cys Tyr 1 5 1015 Glu Ala Thr Asp Thr Glu Thr Gly Ser Ala Tyr Ala Val Lys Val Ile 20 2530 Pro Gln Ser Arg Val Ala Lys Pro His Gln Arg Glu Lys Ile Leu Asn 35 4045 Glu Ile Glu Leu His Arg Asp Leu Gln His Arg His Ile Val Arg Phe 50 5560 Ser His His Phe Glu Asp Ala Asp Asn Ile Tyr Ile Phe Leu Glu Leu 65 7075 80 Cys Ser Arg Lys Ser Leu Ala His Ile Trp Lys Ala Arg His Thr Leu 8590 95 Leu Glu Pro Glu Val Arg Tyr Tyr Leu Arg Gln Ile Leu Ser Gly Leu100 105 110 Lys Tyr Leu His Gln Arg Gly Ile Leu His Arg Asp Leu Lys LeuGly 115 120 125 Asn Phe Phe Ile Thr Glu Asn Met Glu Leu Lys Val Gly AspPhe Gly 130 135 140 Leu Ala Ala Arg Leu Glu Pro Pro Glu Gln Arg Lys LysThr Ile Cys 145 150 155 160 Gly Thr Pro Asn Tyr Val Ala Pro Glu Val LeuLeu Arg Gln Gly His 165 170 175 Gly Pro Glu Ala Asp Val Trp Ser Leu GlyCys Val Met Tyr Thr Leu 180 185 190 Leu Cys Gly Ser Pro Pro Phe Glu ThrAla Asp Leu Lys Glu Thr Tyr 195 200 205 Arg Cys Ile Lys Gln Val His TyrThr Leu Pro Ala Ser Leu Ser Leu 210 215 220 Pro Ala Arg Gln Leu Leu AlaAla Ile Leu Arg Ala Ser Pro Arg Asp 225 230 235 240 Arg Pro Ser Ile AspGln Ile Leu Arg His Asp Phe Phe 245 250 44 5 PRT Artificial SequenceDescription of Artificial Sequenceconsensus peptide 44 His Arg Asp LeuLys 1 5 45 5 PRT Artificial Sequence Description of ArtificialSequenceconsensus peptide 45 Asp Phe Gly Leu Ala 1 5 46 4 PRT ArtificialSequence Description of Artificial Sequenceconsensus peptide 46 Ala ProGlu Val 1 47 6 PRT Artificial Sequence Description of ArtificialSequenceconsensus peptide 47 Asp Val Trp Ser Leu Gly 1 5 48 256 PRT Homosapiens serine threonine kinase 2 (STK2, NEK4) 48 Tyr Cys Tyr Leu ArgVal Val Gly Lys Gly Ser Tyr Gly Glu Val Thr 1 5 10 15 Leu Val Lys HisArg Arg Asp Gly Lys Gln Tyr Val Ile Lys Lys Leu 20 25 30 Asn Leu Arg AsnAla Ser Ser Arg Glu Arg Arg Ala Ala Glu Gln Glu 35 40 45 Ala Gln Leu LeuSer Gln Leu Lys His Pro Asn Ile Val Thr Tyr Lys 50 55 60 Glu Ser Trp GluGly Gly Asp Gly Leu Leu Tyr Ile Val Met Gly Phe 65 70 75 80 Cys Glu GlyGly Asp Leu Tyr Arg Lys Leu Lys Glu Gln Lys Gly Gln 85 90 95 Leu Leu ProGlu Asn Gln Val Val Glu Trp Phe Val Gln Ile Ala Met 100 105 110 Ala LeuGln Tyr Leu His Glu Lys His Ile Leu His Arg Asp Leu Lys 115 120 125 ThrGln Asn Val Phe Leu Thr Arg Thr Asn Ile Ile Lys Val Gly Asp 130 135 140Leu Gly Ile Ala Arg Val Leu Glu Asn His Cys Asp Met Ala Ser Thr 145 150155 160 Leu Ile Gly Thr Pro Tyr Tyr Met Ser Pro Glu Leu Phe Ser Asn Lys165 170 175 Pro Tyr Asn Tyr Lys Ser Asp Val Trp Ala Leu Gly Cys Cys ValTyr 180 185 190 Glu Met Ala Thr Leu Lys His Ala Phe Asn Ala Lys Asp MetAsn Ser 195 200 205 Leu Val Tyr Arg Ile Ile Glu Gly Lys Leu Pro Pro MetPro Arg Asp 210 215 220 Tyr Ser Pro Glu Leu Ala Glu Leu Ile Arg Thr MetLeu Ser Lys Arg 225 230 235 240 Pro Glu Glu Arg Pro Ser Val Arg Ser IleLeu Arg Gln Pro Tyr Ile 245 250 255 49 5 PRT Artificial SequenceDescription of Artificial Sequenceconsensus peptide 49 His Pro Asn IleVal 1 5 50 5 PRT Artificial Sequence Description of ArtificialSequenceconsensus peptide 50 Glu Gly Gly Asp Leu 1 5 51 294 PRTArtificial Sequence Description of Artificial Sequenceprotein kinaseconsensus sequence 51 Tyr Glu Leu Leu Glu Lys Leu Gly Glu Gly Ser PheGly Lys Val Tyr 1 5 10 15 Lys Ala Lys His Lys Asp Lys Thr Gly Lys IleVal Ala Val Lys Ile 20 25 30 Leu Lys Lys Glu Lys Glu Ser Ile Lys Glu LysArg Phe Leu Arg Glu 35 40 45 Ile Gln Ile Leu Lys Arg Leu Ser His Pro AsnIle Val Arg Leu Ile 50 55 60 Gly Val Phe Glu Asp Thr Asp Asp His Leu TyrLeu Val Met Glu Tyr 65 70 75 80 Met Glu Gly Gly Asp Leu Phe Asp Tyr LeuArg Arg Asn Gly Gly Pro 85 90 95 Leu Ser Glu Lys Glu Ala Lys Lys Ile AlaLeu Gln Ile Leu Arg Gly 100 105 110 Leu Glu Tyr Leu His Ser Asn Gly IleVal His Arg Asp Leu Lys Pro 115 120 125 Glu Asn Ile Leu Leu Asp Glu AsnAsp Gly Thr Val Lys Ile Ala Asp 130 135 140 Phe Gly Leu Ala Arg Leu LeuGlu Ser Ser Ser Lys Leu Thr Thr Phe 145 150 155 160 Val Gly Thr Pro TrpTyr Met Met Ala Pro Glu Val Ile Leu Glu Gly 165 170 175 Arg Gly Tyr SerSer Lys Val Asp Val Trp Ser Leu Gly Val Ile Leu 180 185 190 Tyr Glu LeuLeu Thr Gly Gly Pro Leu Phe Pro Gly Ala Asp Leu Pro 195 200 205 Ala PheThr Gly Gly Asp Glu Val Asp Gln Leu Ile Ile Phe Val Leu 210 215 220 LysLeu Pro Phe Ser Asp Glu Leu Pro Lys Thr Arg Ile Asp Pro Leu 225 230 235240 Glu Glu Leu Phe Arg Ile Ile Lys Arg Pro Gly Leu Arg Leu Pro Leu 245250 255 Pro Ser Asn Cys Ser Glu Glu Leu Lys Asp Leu Leu Lys Lys Cys Leu260 265 270 Asn Lys Asp Pro Ser Lys Arg Pro Gly Ser Ala Thr Ala Lys GluIle 275 280 285 Leu Asn His Pro Trp Phe 290 52 286 PRT Homo sapiensserine threonine protein kinase casein kinase 2, alpha 1 subunit isoforma, transcript variant 2 (CK2, CK2alpha), CK2 catalytic subunit alpha 52Tyr Gln Leu Val Arg Lys Leu Gly Arg Gly Lys Tyr Ser Glu Val Phe 1 5 1015 Glu Ala Ile Asn Ile Thr Asn Asn Glu Lys Val Val Val Lys Ile Leu 20 2530 Lys Pro Val Lys Lys Lys Lys Ile Lys Arg Glu Ile Lys Ile Leu Glu 35 4045 Asn Leu Arg Gly Gly Pro Asn Ile Ile Thr Leu Ala Asp Ile Val Lys 50 5560 Asp Pro Val Ser Arg Thr Pro Ala Leu Val Phe Glu His Val Asn Asn 65 7075 80 Thr Asp Phe Lys Gln Leu Tyr Gln Thr Leu Thr Asp Tyr Asp Ile Arg 8590 95 Phe Tyr Met Tyr Glu Ile Leu Lys Ala Leu Asp Tyr Cys His Ser Met100 105 110 Gly Ile Met His Arg Asp Val Lys Pro His Asn Val Met Ile AspHis 115 120 125 Glu His Arg Lys Leu Arg Leu Ile Asp Trp Gly Leu Ala GluPhe Tyr 130 135 140 His Pro Gly Gln Glu Tyr Asn Val Arg Val Ala Ser ArgTyr Phe Lys 145 150 155 160 Gly Pro Glu Leu Leu Val Asp Tyr Gln Met TyrAsp Tyr Ser Leu Asp 165 170 175 Met Trp Ser Leu Gly Cys Met Leu Ala SerMet Ile Phe Arg Lys Glu 180 185 190 Pro Phe Phe His Gly His Asp Asn TyrAsp Gln Leu Val Arg Ile Ala 195 200 205 Lys Val Leu Gly Thr Glu Asp LeuTyr Asp Tyr Ile Asp Lys Tyr Asn 210 215 220 Ile Glu Leu Asp Pro Arg PheAsn Asp Ile Leu Gly Arg His Ser Arg 225 230 235 240 Lys Arg Trp Glu ArgPhe Val His Ser Glu Asn Gln His Leu Val Ser 245 250 255 Pro Glu Ala LeuAsp Phe Leu Asp Lys Leu Leu Arg Tyr Asp His Gln 260 265 270 Ser Arg LeuThr Ala Arg Glu Ala Met Glu His Pro Tyr Phe 275 280 285 53 5 PRTArtificial Sequence Description of Artificial Sequenceconsensus peptide53 Val Lys Ile Leu Lys 1 5 54 4 PRT Artificial Sequence Description ofArtificial Sequenceconsensus peptide 54 Trp Ser Leu Gly 1 55 298 PRTHomo sapiens cyclin-dependent kinase 2 (CDK2) 55 Met Glu Asn Phe Gln LysVal Glu Lys Ile Gly Glu Gly Thr Tyr Gly 1 5 10 15 Val Val Tyr Lys AlaArg Asn Lys Leu Thr Gly Glu Val Val Ala Leu 20 25 30 Lys Lys Ile Arg LeuAsp Thr Glu Thr Glu Gly Val Pro Ser Thr Ala 35 40 45 Ile Arg Glu Ile SerLeu Leu Lys Glu Leu Asn His Pro Asn Ile Val 50 55 60 Lys Leu Leu Asp ValIle His Thr Glu Asn Lys Leu Tyr Leu Val Phe 65 70 75 80 Glu Phe Leu HisGln Asp Leu Lys Lys Phe Met Asp Ala Ser Ala Leu 85 90 95 Thr Gly Ile ProLeu Pro Leu Ile Lys Ser Tyr Leu Phe Gln Leu Leu 100 105 110 Gln Gly LeuAla Phe Cys His Ser His Arg Val Leu His Arg Asp Leu 115 120 125 Lys ProGln Asn Leu Leu Ile Asn Thr Glu Gly Ala Ile Lys Leu Ala 130 135 140 AspPhe Gly Leu Ala Arg Ala Phe Gly Val Pro Val Arg Thr Tyr Thr 145 150 155160 His Glu Val Val Thr Leu Trp Tyr Arg Ala Pro Glu Ile Leu Leu Gly 165170 175 Cys Lys Tyr Tyr Ser Thr Ala Val Asp Ile Trp Ser Leu Gly Cys Ile180 185 190 Phe Ala Glu Met Val Thr Arg Arg Ala Leu Phe Pro Gly Asp SerGlu 195 200 205 Ile Asp Gln Leu Phe Arg Ile Phe Arg Thr Leu Gly Thr ProAsp Glu 210 215 220 Val Val Trp Pro Gly Val Thr Ser Met Pro Asp Tyr LysPro Ser Phe 225 230 235 240 Pro Lys Trp Ala Arg Gln Asp Phe Ser Lys ValVal Pro Pro Leu Asp 245 250 255 Glu Asp Gly Arg Ser Leu Leu Ser Gln MetLeu His Tyr Asp Pro Asn 260 265 270 Lys Arg Ile Ser Ala Lys Ala Ala LeuAla His Pro Phe Phe Gln Asp 275 280 285 Val Thr Lys Pro Val Pro His LeuArg Leu 290 295 56 111 PRT Artificial Sequence Description of ArtificialSequenceXeroderma pigmentosum complementation group XPG N- terminaldomain (XPG_N) consensus sequence 56 Met Gly Ile Lys Gly Leu Leu Pro IleLeu Lys Pro Val Ala Pro Glu 1 5 10 15 Ala Ile Arg Ser Val Ser Ile GluAla Leu Glu Gly Tyr Tyr Lys Val 20 25 30 Leu Ala Ile Asp Ala Ser Ile TrpLeu Tyr Gln Phe Leu Lys Ala Val 35 40 45 Arg Asp Gln Leu Gly Asn Asn LeuGlu Asn Glu Glu Gly Glu Thr Thr 50 55 60 Ser His Leu Met Gly Leu Phe SerArg Leu Cys Arg Leu Leu Asp Phe 65 70 75 80 Gly Ile Lys Pro Ile Phe ValPhe Asp Gly Gly Ala Pro Asn Asp Leu 85 90 95 Lys Ala Glu Thr Leu Gln LysArg Ser Ala Arg Arg Gln Glu Ala 100 105 110 57 107 PRT ArtificialSequence flap structure-specific endonuclease 1 (FEN1) 5′-3′ exonuclease57 Met Gly Ile Gln Gly Leu Ala Lys Leu Ile Ala Asp Val Ala Pro Ser 1 510 15 Ala Ile Arg Glu Asn Asp Ile Lys Ser Tyr Phe Gly Arg Lys Val Ala 2025 30 Ile Asp Ala Ser Met Ser Ile Tyr Gln Phe Leu Ile Ala Val Arg Gln 3540 45 Gly Gly Asp Val Leu Gln Asn Glu Glu Gly Glu Thr Thr Ser His Leu 5055 60 Met Gly Met Phe Tyr Arg Thr Ile Arg Met Met Glu Asn Gly Ile Lys 6570 75 80 Pro Val Tyr Val Phe Asp Gly Lys Pro Pro Gln Leu Lys Ser Gly Glu85 90 95 Leu Ala Lys Arg Ser Glu Arg Arg Ala Glu Ala 100 105 58 5 PRTArtificial Sequence Description of Artificial Sequenceconsensus peptide58 Ala Ile Asp Ala Ser 1 5 59 4 PRT Artificial Sequence Description ofArtificial Sequenceconsensus peptide 59 Tyr Gln Phe Leu 1 60 12 PRTArtificial Sequence Description of Artificial Sequenceconsensus peptide60 Asn Glu Glu Gly Glu Thr Thr Ser His Leu Met Gly 1 5 10 61 4 PRTArtificial Sequence Description of Artificial Sequenceconsensus peptide61 Gly Ile Lys Pro 1 62 4 PRT Artificial Sequence Description ofArtificial Sequenceconsensus peptide 62 Val Phe Asp Gly 1 63 104 PRTArtificial Sequence Description of Artificial SequenceXerodermapigmentosum complementation group XPG I-region domain (XPG_I) consensussequence 63 Arg Leu Met Gly Ile Pro Tyr Ile Val Ala Pro Gly Val Glu AlaGlu 1 5 10 15 Ala Gln Cys Ala Tyr Leu Glu Lys Lys Gly Leu Val Asp GlyIle Ile 20 25 30 Thr Glu Asp Ser Asp Val Leu Leu Phe Gly Ala Pro Arg LeuLeu Arg 35 40 45 Asn Leu Thr Leu Ser Gly Lys Lys Ser Gly Pro Ser Ile ThrSer Leu 50 55 60 Lys Val Glu Ile Glu Glu Ile Asp Leu Glu Ser Leu Leu ArgGlu Leu 65 70 75 80 Gly Leu Gly Lys Leu Ser Arg Glu Gln Leu Ile Asp LeuAla Ile Leu 85 90 95 Leu Gly Cys Asp Tyr Thr Glu Gly 100 64 92 PRT Homosapiens flap structure-specific endonuclease 1 (FEN1) 5′-3′ exonuclease64 Ser Leu Met Gly Ile Pro Tyr Leu Asp Ala Pro Ser Glu Ala Glu Ala 1 510 15 Ser Cys Ala Ala Leu Val Lys Ala Gly Lys Val Tyr Ala Ala Ala Thr 2025 30 Glu Asp Met Asp Cys Leu Thr Phe Gly Ser Pro Val Leu Met Arg His 3540 45 Leu Thr Ala Ser Glu Ala Lys Lys Leu Pro Ile Gln Glu Phe His Leu 5055 60 Ser Arg Ile Leu Gln Glu Leu Gly Leu Asn Gln Glu Gln Phe Val Asp 6570 75 80 Leu Cys Ile Leu Leu Gly Ser Asp Tyr Cys Glu Ser 85 90 65 6 PRTArtificial Sequence Description of Artificial Sequenceconsensus peptide65 Leu Met Gly Ile Pro Tyr 1 5 66 4 PRT Artificial Sequence Descriptionof Artificial Sequenceconsensus peptide 66 Glu Ala Glu Ala 1 67 4 PRTArtificial Sequence Description of Artificial Sequenceconsensus peptide67 Glu Leu Gly Leu 1 68 4 PRT Artificial Sequence Description ofArtificial Sequenceconsensus peptide 68 Ile Leu Leu Gly 1 69 261 PRTHomo sapiens HBO1 histone acetyltransferase, MYST histoneacetyltransferase 2 (MYST2) 69 Tyr His Ser Pro Tyr Pro Glu Glu Tyr AlaArg Leu Gly Arg Leu Tyr 1 5 10 15 Met Cys Glu Phe Cys Leu Lys Tyr MetLys Ser Gln Thr Ile Leu Arg 20 25 30 Arg His Met Ala Lys Cys Val Trp LysHis Pro Pro Gly Asp Glu Ile 35 40 45 Tyr Arg Lys Gly Ser Ile Ser Val PheGlu Val Asp Gly Lys Lys Asn 50 55 60 Lys Ile Tyr Cys Gln Asn Leu Cys LeuLeu Ala Lys Leu Phe Leu Asp 65 70 75 80 His Lys Thr Leu Tyr Tyr Asp ValGlu Pro Phe Leu Phe Tyr Val Met 85 90 95 Thr Glu Ala Asp Asn Thr Gly CysHis Leu Ile Gly Tyr Phe Ser Lys 100 105 110 Glu Lys Asn Ser Phe Leu AsnTyr Asn Val Ser Cys Ile Leu Thr Met 115 120 125 Pro Gln Tyr Met Arg GlnGly Tyr Gly Lys Met Leu Ile Asp Phe Ser 130 135 140 Tyr Leu Leu Ser LysVal Glu Glu Lys Val Gly Ser Pro Glu Arg Pro 145 150 155 160 Leu Ser AspLeu Gly Leu Ile Ser Tyr Arg Ser Tyr Trp Lys Glu Val 165 170 175 Leu LeuArg Tyr Leu His Asn Phe Gln Gly Lys Glu Ile Ser Ile Lys 180 185 190 GluIle Ser Gln Glu Thr Ala Val Asn Pro Val Asp Ile Val Ser Thr 195 200 205Leu Gln Ala Leu Gln Met Leu Lys Tyr Trp Lys Gly Lys His Leu Val 210 215220 Leu Lys Arg Gln Asp Leu Ile Asp Glu Trp Ile Ala Lys Glu Ala Lys 225230 235 240 Arg Ser Asn Ser Asn Lys Thr Met Asp Pro Ser Cys Leu Lys TrpThr 245 250 255 Pro Pro Lys Gly Thr 260 70 265 PRT Saccharomycescerevisiae Esa1 70 Tyr Phe Ser Pro Tyr Pro Ile Glu Leu Thr Asp Glu AspPhe Ile Tyr 1 5 10 15 Ile Asp Asp Phe Thr Leu Gln Tyr Phe Gly Ser LysLys Gln Tyr Glu 20 25 30 Arg Tyr Arg Lys Lys Cys Thr Leu Arg His Pro ProGly Asn Glu Ile 35 40 45 Tyr Arg Asp Asp Tyr Val Ser Phe Phe Glu Ile AspGly Arg Lys Gln 50 55 60 Arg Thr Trp Cys Arg Asn Leu Cys Leu Leu Ser LysLeu Phe Leu Asp 65 70 75 80 His Lys Thr Leu Tyr Tyr Asp Val Asp Pro PheLeu Phe Tyr Cys Met 85 90 95 Thr Arg Arg Asp Glu Leu Gly His His Leu ValGly Tyr Phe Ser Lys 100 105 110 Glu Lys Glu Ser Ala Asp Gly Tyr Asn ValAla Cys Ile Leu Thr Leu 115 120 125 Pro Gln Tyr Gln Arg Met Gly Tyr GlyLys Leu Leu Ile Glu Phe Ser 130 135 140 Tyr Glu Leu Ser Lys Lys Glu AsnLys Val Gly Ser Pro Glu Lys Pro 145 150 155 160 Leu Ser Asp Leu Gly LeuLeu Ser Tyr Arg Ala Tyr Trp Ser Asp Thr 165 170 175 Leu Ile Thr Leu LeuVal Glu His Gln Lys Glu Ile Thr Ile Asp Glu 180 185 190 Ile Ser Ser MetThr Ser Met Thr Thr Thr Asp Ile Leu His Thr Ala 195 200 205 Lys Thr LeuAsn Ile Leu Arg Tyr Tyr Lys Gly Gln His Ile Ile Phe 210 215 220 Leu AsnGlu Asp Ile Leu Asp Arg Tyr Asn Arg Leu Lys Ala Lys Lys 225 230 235 240Arg Arg Thr Ile Asp Pro Asn Arg Leu Ile Trp Lys Pro Pro Val Phe 245 250255 Thr Ala Ser Gln Leu Arg Phe Ala Trp 260 265 71 253 PRT Homo sapiensPIM1 oncogene serine threonine kinase 71 Tyr Gln Val Gly Pro Leu Leu GlySer Gly Gly Phe Gly Ser Val Tyr 1 5 10 15 Ser Gly Ile Arg Val Ser AspAsn Leu Pro Val Ala Ile Lys His Val 20 25 30 Glu Lys Asp Arg Ile Ser AspTrp Gly Glu Leu Pro Asn Gly Thr Arg 35 40 45 Val Pro Met Glu Val Val LeuLeu Lys Lys Val Ser Ser Gly Phe Ser 50 55 60 Gly Val Ile Arg Leu Leu AspTrp Phe Glu Arg Pro Asp Ser Phe Val 65 70 75 80 Leu Ile Leu Glu Arg ProGlu Pro Val Gln Asp Leu Phe Asp Phe Ile 85 90 95 Thr Glu Arg Gly Ala LeuGln Glu Glu Leu Ala Arg Ser Phe Phe Trp 100 105 110 Gln Val Leu Glu AlaVal Arg His Cys His Asn Cys Gly Val Leu His 115 120 125 Arg Asp Ile LysAsp Glu Asn Ile Leu Ile Asp Leu Asn Arg Gly Glu 130 135 140 Leu Lys LeuIle Asp Phe Gly Ser Gly Ala Leu Leu Lys Asp Thr Val 145 150 155 160 TyrThr Asp Phe Asp Gly Thr Arg Val Tyr Ser Pro Pro Glu Trp Ile 165 170 175Arg Tyr His Arg Tyr His Gly Arg Ser Ala Ala Val Trp Ser Leu Gly 180 185190 Ile Leu Leu Tyr Asp Met Val Cys Gly Asp Ile Pro Phe Glu His Asp 195200 205 Glu Glu Ile Ile Arg Gly Gln Val Phe Phe Arg Gln Arg Val Ser Ser210 215 220 Glu Cys Gln His Leu Ile Arg Trp Cys Leu Ala Leu Arg Pro SerAsp 225 230 235 240 Arg Pro Thr Phe Glu Glu Ile Gln Asn His Pro Trp Met245 250 72 4 PRT Artificial Sequence Description of ArtificialSequenceconsensus peptide 72 Asp Leu Phe Asp 1 73 4 PRT ArtificialSequence Description of Artificial Sequenceconsensus peptide 73 Glu AsnIle Leu 1 74 5 PRT Artificial Sequence Description of ArtificialSequenceconsensus peptide 74 Val Trp Ser Leu Gly 1 5 75 4 PRT ArtificialSequence Description of Artificial Sequenceconsensus peptide 75 Asn HisPro Trp 1 76 13 DNA Artificial Sequence Description of ArtificialSequence5′-end 32P-labeled oligonucleotide primer 76 cactgactgt atg 1377 30 DNA/RNA Artificial Sequence Description of Combined DNA/RNAMoleculeoligonucleotide template 77 ctcgtcagca tcttcaucat acagtcagtg 3078 200 PRT Artificial Sequence Description of Artificial SequencepolyGly flexible linker 78 Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly GlyGly Gly Gly Gly 1 5 10 15 Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly GlyGly Gly Gly Gly Gly 20 25 30 Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly GlyGly Gly Gly Gly Gly 35 40 45 Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly GlyGly Gly Gly Gly Gly 50 55 60 Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly GlyGly Gly Gly Gly Gly 65 70 75 80 Gly Gly Gly Gly Gly Gly Gly Gly Gly GlyGly Gly Gly Gly Gly Gly 85 90 95 Gly Gly Gly Gly Gly Gly Gly Gly Gly GlyGly Gly Gly Gly Gly Gly 100 105 110 Gly Gly Gly Gly Gly Gly Gly Gly GlyGly Gly Gly Gly Gly Gly Gly 115 120 125 Gly Gly Gly Gly Gly Gly Gly GlyGly Gly Gly Gly Gly Gly Gly Gly 130 135 140 Gly Gly Gly Gly Gly Gly GlyGly Gly Gly Gly Gly Gly Gly Gly Gly 145 150 155 160 Gly Gly Gly Gly GlyGly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly 165 170 175 Gly Gly Gly GlyGly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly 180 185 190 Gly Gly GlyGly Gly Gly Gly Gly 195 200

What is claimed is:
 1. A method for identifying a compound thatmodulates cell cycle arrest, the method comprising the steps of: (i)contacting a cell comprising a target polypeptide or fragment thereof orinactive variant thereof, selected from the group consisting of flapstructure specific endonuclease 1 (FEN1), protein kinase C ζ (PKC-ζ),phospholipase C-β1 (PLC-β1), protein tyrosine kinase 2 (FAK), proteintyrosine kinase 2b (FAK2), casein kinase 2 (CK2), cMET tyrosine kinase(cMET), REV1 dCMP transferase (REV 1), apurinic/apyrimidinic nuclease 1(APE1), cyclin dependent kinase 3 (CDK3), PIM1 kinase (PIM1), celldivision cycle 7 kinase (CDC7L1), cyclin dependent kinase 7 (CDK7),cytokine inducible kinase (CNK), potentially prenylated protein tyrosinephosphatase (PRL-3), serine threonine kinase 2 (STK2) or (NEK4), cyclindependent serine threonine kinase (NKIAMRE), or histone acetylase(HBO1), or fragment thereof with the compound, the target polypeptideencoded by the complement of a nucleic acid that hybridizes understringent conditions to a nucleic acid encoding a polypeptide having anamino acid sequence selected from the group consisting of SEQ ID NO:14,2, 4, 6, 8, 10, 12, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, and 36; and(ii) determining the chemical or phenotypic effect of the compound uponthe cell comprising the target polypeptide or fragment thereof orinactive variant thereof, thereby identifying a compound that modulatescell cycle arrest.
 2. The method of claim 1, wherein the chemical orphenotypic effect is determined by measuring enzymatic activity selectedfrom the group consisting of nuclease activity, kinase activity, lipaseactivity, transferase activity, phosphatase activity, and acetylaseactivity.
 3. The method of claim 1, wherein the chemical or phenotypiceffect is determined by measuring cellular proliferation.
 4. The methodof claim 3, wherein the cellular proliferation is measured by assayingfluorescent marker level or DNA synthesis.
 5. The method of claim 4,wherein DNA synthesis is measured by ³H thymidine incorporation, BrdUincorporation, or Hoescht staining.
 6. The method of claim 4, whereinthe fluorescent marker is selected from the group consisting of a celltracker dye or green fluorescent protein.
 7. The method of claim 1,wherein modulation is activation of cell cycle arrest.
 8. The method ofclaim 1, wherein modulation is activation of cancer cell cycle arrest.9. The method of claim 1, wherein the host cell is a cancer cell. 10.The method of claim 9, wherein the cancer cell is a breast, prostate,colon, or lung cancer cell.
 11. The method of claim 9, wherein thecancer cell is a transformed cell line.
 12. The method of claim 11,wherein the transformed cell line is A549, PC3, H1299, MDA-MB-231, MCF7,or HeLa.
 13. The method of claim 9, wherein the cancer cell is p53 nullor mutant.
 14. The method of claim 9, wherein the cancer cell is p53wild-type.
 15. The method of claim 1, wherein the polypeptide isrecombinant.
 16. The method of claim 1, wherein the polypeptide isencoded by a nucleic acid comprising a sequence of SEQ ID NO:13, 1, 3,5, 7, 9, 11, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, or
 35. 17. Themethod of claim 1, wherein the compound is an antibody.
 18. The methodof claim 1, wherein the compound is a small organic molecule.
 19. Themethod of claim 1, wherein the compound is an antisense molecule. 20.The method of claim 1, wherein the compound is a peptide.
 21. The methodof claim 20, wherein the peptide is circular.
 22. The method of claim 1,wherein the compound is an siRNA molecule.
 23. A method for identifyinga compound that modulates cell cycle arrest, the method comprising thesteps of: (i) contacting a cell comprising a target polypeptide orfragment thereof or inactive variant thereof, selected from the groupconsisting of flap structure specific endonuclease 1 (FEN1), proteinkinase C ζ (PKC-ζ), phospholipase C-β1 (PLC-β1), protein tyrosine kinase2 (FAK), protein tyrosine kinase 2b (FAK2), casein kinase 2 (CK2), cMETtyrosine kinase (cMET), REV1 dCMP transferase (REV 1),apurinic/apyrimidinic nuclease 1 (APE1), cyclin dependent kinase 3(CDK3), PIM1 kinase (PIM1), cell division cycle 7 kinase (CDC7L1),cyclin dependent kinase 7 (CDK7), cytokine inducible kinase (CNK),potentially prenylated protein tyrosine phosphatase (PRL-3), serinethreonine kinase 2 (STK2) or (NEK4), cyclin dependent serine threoninekinase (NKIAMRE), or histone acetylase (HBO1), or fragment thereof withthe compound, the target polypeptide encoded by the complement of anucleic acid that hybridizes under stringent conditions to a nucleicacid encoding a polypeptide having an amino acid sequence selected fromthe group consisting of SEQ ID NO:14, 2, 4, 6, 8, 10, 12, 16, 18, 20,22, 24, 26, 28, 30, 32, 34, and 36; and (ii) determining the physicaleffect of the compound upon the target polypeptide or fragment thereofor inactive variant thereof; and (iii) determining the chemical orphenotypic effect of the compound upon a cell comprising the targetpolypeptide or or fragment thereof or inactive variant thereof, therebyidentifying a compound that modulates cell cycle arrest.
 24. A method ofmodulating cell cycle arrest in a subject, the method comprising thestep of administering to the subject a therapeutically effective amountof a compound identified using the method of claim
 1. 25. The method ofclaim 24, wherein the subject is a human.
 26. The method of claim 25,wherein the subject has cancer.
 27. The method of claim 24, wherein thecompound is a small organic molecule.
 28. The method of claim 24,wherein the compound is an antisense molecule.
 29. The method of claim24, wherein the compound is an antibody.
 30. The method of claim 24,wherein the compound is a peptide.
 31. The method of claim 30, whereinthe peptide is circular.
 32. The method of claim 24, wherein thecompound is an siRNA molecule.
 33. The method of claim 24, wherein thecompound inhibits cancer cell proliferation.
 34. A method of modulatingcell cycle arrests in a subject, the method comprising the step ofadministering to the subject a therapeutically effective amount of atarget polypeptide or fragment thereof or inactive variant thereof,selected from the group consisting of flap structure specificendonuclease 1 (FEN1), protein kinase C ζ (PKC-ζ), phospholipase C-β1(PLC-β1), protein tyrosine kinase 2 (FAK), protein tyrosine kinase 2b(FAK2), casein kinase 2 (CK2), cMET tyrosine kinase (cMET), REV1 dCMPtransferase (REV 1), apurinic/apyrimidinic nuclease 1 (APE1), cyclindependent kinase 3 (CDK3), PIM1 kinase (PIM1), cell division cycle 7kinase (CDC7L1), cyclin dependent kinase 7 (CDK7), cytokine induciblekinase (CNK), potentially prenylated protein tyrosine phosphatase(PRL-3), serine threonine kinase 2 (STK2) or (NEK4), cyclin dependentserine threonine kinase (NKIAMRE), or histone acetylase (HBO1), orfragment thereof with the compound, the target polypeptide encoded bythe complement of a nucleic acid that hybridizes under stringentconditions to a nucleic acid encoding a polypeptide having an amino acidsequence selected from the group consisting of SEQ ID NO:14, 2, 4, 6, 8,10, 12, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, and
 36. 35. A method ofmodulating cell cycle arrest in a subject, the method comprising thestep of administering to the subject a therapeutically effective amountof a nucleic acid encoding a target polypeptide or fragment thereof orinactive variant thereof, selected from the group consisting of flapstructure specific endonuclease 1 (FEN1), protein kinase C ζ(PKC-ζ),phospholipase C-β1 (PLC-β1), protein tyrosine kinase 2 (FAK), proteintyrosine kinase 2b (FAK2), casein kinase 2 (CK2), cMET tyrosine kinase(cMET), REV1 dCMP transferase (REV1), apurinic/apyrimidinic nuclease 1(APE1), cyclin dependent kinase 3 (CDK3), PIM1 kinase (PIM1), celldivision cycle 7 kinase (CDC7L1), cyclin dependent kinase 7 (CDK7),cytokine inducible kinase (CNK), potentially prenylated protein tyrosinephosphatase (PRL-3), serine threonine kinase 2 (STK2) or (NEK4), cyclindependent serine threonine kinase (NKIAMRE), or histone acetylase(HBO1), or fragment thereof with the compound, the target polypeptideencoded by the complement of a nucleic acid that hybridizes understringent conditions to a nucleic acid encoding a polypeptide having anamino acid sequence selected from the group consisting of SEQ ID NO:14,2, 4, 6, 8, 10, 12, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, and
 36. 36.A CK2-specific siRNA molecule comprising the sequenceAACATTGAATTAGATCCACGT, wherein the siRNA molecule is from 21 to 30nucleotide base pairs in length.
 37. The CK2-specific siRNA molecule ofclaim 36 consisting of the sequence AACATTGAATTAGATCCACGT and itscomplement as active portion.
 38. A method of inhibiting expression of aCK2 gene in a cell, the method comprising contacting the cell with aCK2-specific siRNA molecule comprising the sequenceAACATTGAATTAGATCCACGT, wherein the siRNA molecule is from 21 to 30nucleotide base pairs in length.
 39. A PIM1-specific siRNA moleculecomprising the sequence AAAACTCCGAGTGAACTGGTC, wherein the siRNAmolecule is from 21 to 30 nucleotide base pairs in length.
 40. ThePIM1-specific siRNA molecule of claim 39 consisting of the sequenceAAAACTCCGAGTGAACTGGTC and its complement as active portion.
 41. A methodof inhibiting expression of a PIM1 gene in a cell, the method comprisingcontacting the cell with a PIM1-specific siRNA molecule comprising thesequence AAAACTCCGAGTGAACTGGTC, wherein the siRNA molecule is from 21 to30 nucleotide base pairs in length.
 42. An Hbo1-specific siRNA moleculecomprising the sequence AACTGAGCAAGTGGTTGATTT, wherein the siRNAmolecule is from 21 to 30 nucleotide base pairs in length.
 43. The Hbo1-specific siRNA molecule of claim 42 consisting of the sequenceAACTGAGCAAGTGGTTGATTT and its complement as active portion.
 44. A methodof inhibiting expression of an Hbo 1 gene in a cell, the methodcomprising contacting the cell with an Hbo1-specific siRNA moleculecomprising the sequence AACTGAGCAAGTGGTTGATTT, wherein the siRNAmolecule is from 21 to 30 nucleotide base pairs in length.